Production of high mannose proteins in plant culture

ABSTRACT

A device, system and method for producing glycosylated proteins in plant culture, particularly proteins having a high mannose glycosylation, while targeting such proteins with an ER signal and/or by-passing the Golgi. The invention further relates to vectors and methods for expression and production of enzymatically active high mannose lysosomal enzymes using transgenic plant root, particularly carrot cells. More particularly, the invention relates to host cells, particularly transgenic suspended carrot cells, vectors and methods for high yield expression and production of biologically active high mannose Glucocerebrosidase (GCD). The invention further provides for compositions and methods for the treatment of lysosomal storage diseases.

RELATED APPLICATIONS

This Application is a Divisional of pending U.S. patent application Ser.No. 11/790,991 filed on Apr. 30, 2007, which is a continuation-in-partof U.S. patent application Ser. No. 10/554,387 filed on Oct. 25, 2005,which is a National Phase of IL2004/000181 filed on Feb. 24, 2004, whichclaims the benefit of Israel Patent Application No. 155588, filed Apr.27, 2003. The contents of the above Applications are all incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to transformed host cells for theproduction of high mannose proteins and a method and system forproducing these proteins, particularly in plant culture.

BACKGROUND OF THE INVENTION

Gaucher's disease is the most prevalent lysosomal storage disorder. Itis caused by a recessive genetic disorder (chromosome 1 q21-q31)resulting in deficiency of glucocerebrosidase, also known asglucosylceramidase, which is a membrane-bound lysosomal enzyme thatcatalyzes the hydrolysis of the glycosphingolipid glucocerebroside(glucosylceramide, GlcCer) to glucose and ceramide. Gaucher disease iscaused by point mutations in the hGCD (human glucocerebrosidase) gene(GBA), which result in accumulation of GlcCer in the lysosomes ofmacrophages. The characteristic storage cells, called Gaucher cells, arefound in liver, spleen and bone marrow. The associated clinical symptomsinclude severe hepatosplenomegaly, anemia, thrombocytopenia and skeletaldeterioration.

The gene encoding human GCD was first sequenced in 1985 (6) The proteinconsists of 497 amino acids derived from a 536-mer pro-peptide. Themature hGCD contains five N-glycosylation amino acid consensus sequences(Asn-X-Ser/Thr). Four of these sites are normally glycosylated.Glycosylation of the first site is essential for the production ofactive protein. Both high-mannose and complex oligosaccharide chainshave been identified (7). hGCD from placenta contains 7% carbohydrate,20% of which is of the high-mannose type (8). Biochemical andsite-directed mutagenesis studies have provided an initial map ofregions and residues important to folding, activator interaction, andactive site location (9).

Treatment of placental hGCD with neuraminidase (yielding an asialoenzyme) results in increased clearance and uptake rates by rat livercells with a concomitant increase in hepatic enzymatic activity (Furbishet al., 1981, Biochim. Biophys. Acta 673:425-434). This glycan-modifiedplacental hGC is currently used as a therapeutic agent in the treatmentof Gaucher's disease. Biochemical and site-directed mutagenesis studieshave provided an initial map of regions and residues important tofolding, activator interaction, and active site location [Grace et al.,J. Biol. Chem. 269:2283-2291 (1994)].

There are three different types of Gaucher disease, each determined bythe level of hGC activity. The major cells affected by the disease arethe macrophages, which are highly enlarged due to GlcCer accumulation,and are thus referred to as “Gaucher cells”.

The identification of a defect in GCD as the primary cause of Gaucher'sdisease led to the development of enzyme replacement therapy as atherapeutic strategy for this disorder.

De Duve first suggested that replacement of the missing lysosomal enzymewith exogenous biologically active enzyme might be a viable approach totreatment of lysosomal storage diseases [Fed Proc. 23:1045 (1964)].

Since that time, various studies have suggested that enzyme replacementtherapy may be beneficial for treating various lysosomal storagediseases. The best success has been shown with individuals with type IGaucher disease, who were treated with exogenous enzyme(β-glucocerebrosidase), prepared from placenta (Ceredase™) or, morerecently, recombinantly (Cerezyme™).

Unmodified glucocerebrosidase derived from natural sources is aglycoprotein with four carbohydrate chains. This protein does not targetthe phagocytic cells in the body and is therefore of limited therapeuticvalue. In developing the current therapy for Gaucher's disease, theterminal sugars on the carbohydrate chains of glucocerebrosidase aresequentially removed by treatment with three different glycosidases.This glycosidase treatment results in a glycoprotein whose terminalsugars consist of mannose residues. Since phagocytes have mannosereceptors that recognize glycoproteins and glycopeptides witholigosaccharide chains that terminate in mannose residues, thecarbohydrate remodeling of glucocerebrosidase has improved the targetingof the enzyme to these cells [Furbish et al., Biochem. Biophys. Acta673:425, (1981)].

As indicated herein, glycosylation plays a crucial role in hGCDactivity, therefore deglycosylation of hGCD expressed in cell linesusing either tunicamycin (Sf9 cells) or point mutations abolishing allglycosylation sites (both Sf9 and COS-1 cells), results in complete lossof enzymatic activity. In addition, hGCD expressed in E. coli was foundto be inactive. Further research indicated the significance of thevarious glycosylation sites for protein activity. In addition to therole of glycosylation in the actual protein activity, the commerciallyproduced enzyme contains glycan sequence modifications that facilitatespecific drug delivery. The glycosylated proteins are remodeledfollowing extraction to include only mannose containing glycansequences.

The human GCD enzyme contains 4 glycosylation sites and 22 lysines. Therecombinantly produced enzyme (Cerezyme™) differs from the placentalenzyme (Ceredase™) in position 495 where an arginine has beensubstituted with a histidine. Furthermore, the oligosaccharidecomposition differs between the recombinant and the placental GCD as theformer has more fucose and N-acetyl-glucosamine residues while thelatter retains one high mannose chain. As mentioned above, both types ofGCDs are treated with three different glycosidases (neuraminidase,galactosidase, and P—N acetyl-glucosaminidase) to expose terminalmannoses, which enables targeting of phagocytic cells. A pharmaceuticalpreparation comprising the recombinantly produced enzyme is described inU.S. Pat. No. 5,549,892. It should be noted that all referencesmentioned are hereby incorporated by reference as if fully set forthherein.

One drawback associated with existing lysosomal enzyme replacementtherapy treatment is that the in vivo bioactivity of the enzyme isundesirably low, e.g. because of low uptake, reduced targeting tolysosomes of the specific cells where the substrate is accumulated, anda short functional in vivo half-life in the lysosomes.

Another major drawback of the existing GCD recombinant enzymes is theirexpense, which can place a heavy economic burden on health care systems.The high cost of these recombinant enzymes results from a complexpurification protocol, and the relatively large amounts of thetherapeutic required for existing treatments. There is therefore, anurgent need to reduce the cost of GCD so that this life saving therapycan be provided to all who require it more affordably.

Proteins for pharmaceutical use have been traditionally produced inmammalian or bacterial expression systems. In the past decade a newexpression system has been developed in plants. This methodologyutilizes Agrobacterium, a bacteria capable of inserting single strandedDNA molecules (T-DNA) into the plant genome. Due to the relativesimplicity of introducing genes for mass production of proteins andpeptides, this methodology is becoming increasingly popular as analternative protein expression system (1).

While post translational modifications do not exist in bacterialexpression systems, plant derived expression systems do facilitate thesemodifications known to be crucial for protein expression and activity.One of the major differences between mammalian and plant proteinexpression system is the variation of protein sugar side chains, causedby the differences in biosynthetic pathways. Glycosylation was shown tohave a profound effect on activity, folding, stability, solubility,susceptibility to proteases, blood clearance rate and antigenicpotential of proteins. Hence, any protein production in plants shouldtake into consideration the potential ramifications of plantglycosylation.

Protein glycosylation is divided into two categories: N-linked andO-linked modifications (2). The two types differ in amino acid to whichthe glycan moiety is attached to—N-linked are attached to Asn residues,while O-linked are attached to Ser or Thr residues. In addition, theglycan sequences of each type bears unique distinguishing features. Ofthe two types, N-linked glycosylation is the more abundant, and itseffect on protein function has been extensively studied. O-linkedglycans, on the other hand are relatively scarce, and less informationis available regarding their affect on proteins.

SUMMARY OF THE INVENTION

The background art does not teach or suggest a device, system or methodfor selectively producing glycosylated proteins in plant culture. Thebackground art also does not teach or suggest such a device, system ormethod for producing high mannose proteins in plant culture. Thebackground art also does not teach or suggest a device, system or methodfor producing proteins in plant culture through the endoplasmicreticulum (ER). The background art also does not teach or suggest such adevice, system or method for producing proteins in plant culture throughthe endoplasmic reticulum (ER) while by-passing the Golgi body. Thebackground art also does not teach or suggest such a device, system ormethod for producing proteins in plant culture by using an ER signal toby-pass the Golgi body.

The present invention overcomes these disadvantages of the backgroundart by providing a device, system and method for producing glycosylatedproteins in plant culture, particularly proteins having a high mannoseglycosylation, while optionally and preferably targeting (and/orotherwise manipulating processing of) such proteins with an ER signal.Without wishing to be limited by a single hypothesis, it is believedthat such targeting causes the proteins to by-pass the Golgi body andthereby to retain the desired glycosylation, particularly high mannoseglycosylation. It should be noted that the term “plant culture” as usedherein includes any type of transgenic and/or otherwise geneticallyengineered plant cell that is grown in culture. The genetic engineeringmay optionally be permanent or transient. Preferably, the culturefeatures cells that are not assembled to form a complete plant, suchthat at least one biological structure of a plant is not present.Optionally and preferably, the culture may feature a plurality ofdifferent types of plant cells, but preferably the culture features aparticular type of plant cell. It should be noted that optionally plantcultures featuring a particular type of plant cell may be originallyderived from a plurality of different types of such plant cells.

The plant cells may be grown according to any type of suitable culturingmethod, including but not limited to, culture on a solid surface (suchas a plastic culturing vessel or plate for example) or in suspension.

The invention further relates to vectors and methods for expression andproduction of enzymatically active high mannose lysosomal enzymes usingtransgenic plant root, particularly carrot cells. More particularly, theinvention relates to host cells, particularly transgenic suspendedcarrot cells, vectors and methods for high yield expression andproduction of biologically active high mannose Glucocerebrosidase (GCD).The invention further provides for compositions and methods for thetreatment of lysosomal storage diseases.

The present invention is also of a device, system and method forproviding sufficient quantities of biologically active lysosomalenzymes, and particularly, human GCD, to deficient cells. The presentinvention is also of host cells comprising new vector compositions thatallow for efficient production of genes encoding lysosomal enzymes, suchas GCD.

The present invention therefore solves a long-felt need for aneconomically viable technology to produce proteins having particularglycosylation requirements, such as the high mannose glycosylation oflysosomal enzymes such as GCD for example. The present invention is ableto solve this long felt need by using plant cell culture.

In order to further explain the present invention, a brief explanationis now provided of the biosynthetic pathway of high-mannose proteins.The basic biosynthesis pathway of high-mannose and complex N-linkedglycans is highly conserved among all eukaryotes. Biosynthesis begins inthe Endoplasmic Reticulum (ER) with the transfer of the glycan precursorfrom a dolichol lipid carrier to a specific Asn residue on the proteinby the oligosaccharyl transferase. The precursor is subsequentlymodified in the ER by glycosidases I and II and a hypotheticalmannosidase to yield the high mannose structures, similar to the processoccurring in mammals.

Further modifications of the glycan sequence to complex and hybridstructures occur in the Golgi. Such modifications include removal of oneof the four mannose residues by α-mannosidase I, addition of anN-acetylglucosamine residue, removal of the two additional mannoseresidues by α-mannosidase II, addition of N-acetylglucosamine andoptionally, at this stage, xylose and fucose residues may be added toyield plant specific N-linked glycans. After the transfer of xylose andfucose to the core, complex type N-glycans can be further processed viathe addition of terminal fucose and galactose. Further modifications maytake place during the glycoprotein transport.

Several approaches are currently used in the background art to controland tailor protein glycosylation in plants, all of which havesignificant deficiencies, particularly in comparison to the presentinvention. Gross modifications, such as complete inhibition ofglycosylation or the removal of glycosylation sites from the peptidechain is one strategy. However, this approach can result in structuraldefects. An additional approach involves knock-out and introduction ofspecific carbohydrate processing enzymes. Again, this approach isdifficult and may also have detrimental effects on the plant cellsthemselves.

The present invention overcomes these deficiencies of the background artapproaches by using an ER signal and/or by blocking secretion from theER to the Golgi body. Without wishing to be limited by a singlehypothesis, since a high mannose structure of lysosomal enzymes ispreferred, if secretion can be blocked and the protein can be maintainedin the ER, naturally occurring high mannose structures are obtainedwithout the need for remodeling.

As indicated above, proteins transported via the endomembrane systemfirst pass into the endoplasmic reticulum. The necessary transportsignal for this step is represented by a signal sequence at theN-terminal end of the molecule, the so-called signal peptide. As soon asthis signal peptide has fulfilled its function, which is to insert theprecursor protein attached to it into the endoplasmic reticulum, it issplit off proteolytically from the precursor protein. By virtue of itsspecific function, this type of signal peptide sequence has beenconserved to a high degree during evolution in all living cells,irrespective of whether they are bacteria, yeasts, fungi, animals orplants.

Many plant proteins, which are inserted into the endoplasmic reticulumby virtue of the signal peptide do not reside in the ER, but aretransported from the endoplasmic reticulum to the Golgi and continuetrafficking from the Golgi to the vacuoles. One class of such sortingsignals for this traffic resides are signals that reside on theC-terminal part of the precursor protein [Neuhaus and Rogers, (1998)Plant Mol. Biol. 38:127-144]. Proteins containing both an N-terminalsignal peptide for insertion into the endoplasmic reticulum and aC-terminal vacuolar targeting signal are expected to contain complexglycans, which is attached to them in the Golgi [Lerouge et al., (1998)Plant Mol. Biol. 38:31-48]. The nature of such C-terminal sortingsignals can vary very widely. U.S. Pat. No. 6,054,637 describes peptidefragments obtained from the region of tobacco basic chitinase, which isa vacuolar protein that act as vacuolar targeting peptides. An examplefor a vacuolar protein containing a C-terminal targeting signal andcomplex glycans is the phaseolin storage protein from bean seeds[Frigerio et al., (1998) Plant Cell 10:1031-1042; Frigerio et al.,(2001) Plant Cell 13:1109-1126.].

The paradigm is that in all eukaryotic cells vacuolar proteins pass viathe ER and the Golgi before sequestering in the vacuole as their finaldestination. Surprisingly, the transformed plant root cells of thepresent invention produced an unexpected high mannose GCD.Advantageously, this high mannose product was found to be biologicallyactive and therefore no further steps were needed for its activation.Without wishing to be limited by a single hypothesis, it would appearthat the use of an ER signal with the recombinant protein being producedin plant cell culture was able to overcome transportation to the Golgi,and hence to retain the desired high mannose glycosylation. Optionally,any type of mechanism which is capable to produce high mannoseglycosylation, including any type of mechanism to by-pass the Golgi, maybe used in accordance with the present invention.

In a first aspect, the present invention relates to a host cellproducing a high mannose recombinant protein of interest. This cell maybe transformed or transfected with a recombinant nucleic acid moleculeencoding a protein of interest or with an expression vector comprisingthe nucleic acid molecule. Such nucleic acid molecule comprises a firstnucleic acid sequence encoding the protein of interest operably linkedto a second nucleic acid sequence encoding a vacuolar targeting signalpeptide. The first nucleic acid sequence may be optionally furtheroperably linked to a third nucleic acid sequence encoding an ER(endoplasmic reticulum) targeting signal peptide. The host cell of theinvention is characterized in that the protein of interest is producedby the cell in a highly mannosylated form.

The host cell of the invention may be a eukaryotic or prokaryotic cell.

In one embodiment, the host cell of the invention is a prokaryotic cell,preferably, a bacterial cell, most preferably, an Agrobacteriumtumefaciens cell. These cells are used for infecting the preferred planthost cells described below.

In another preferred embodiment, the host cell of the invention may be aeukaryotic cell, preferably, a plant cell, and most preferably, a plantroot cell selected from the group consisting of Agrobacterium rihzogenestransformed root cell, celery cell, ginger cell, horseradish cell andcarrot cell.

In a preferred embodiment, the plant root cell is a carrot cell. Itshould be noted that the transformed carrot cells of the invention aregrown in suspension. As mentioned above and described in the Examples,these cells were transformed with the Agrobacterium tumefaciens cells.

In another embodiment, the recombinant nucleic acid molecule comprisedwithin the host cell of the invention, comprises a first nucleic acidsequence encoding a lysosomal enzyme that is in operable linkage with asecond nucleic acid sequence encoding a vacuolar targeting signalpeptide derived from the basic tobacco chitinase A gene. This vacuolarsignal peptide has the amino acid sequence as denoted by SEQ ID NO: 2.The first nucleic acid sequence may be optionally further linked in anoperable linkage with a third nucleic acid sequence encoding an ER(endoplasmic reticulum) targeting signal peptide as denoted by SEQ IDNO: 1. In one embodiment, the recombinant nucleic acid moleculecomprised within the host cell of the invention further comprises apromoter that is functional in plant cells. This promoter should beoperably linked to the recombinant molecule of the invention.

In another embodiment, this recombinant nucleic acid molecule mayoptionally further comprise an operably linked terminator which ispreferably functional in plant cells. The recombinant nucleic acidmolecule of the invention may optionally further comprise additionalcontrol, promoting and regulatory elements and/or selectable markers. Itshould be noted that these regulatory elements are operably linked tothe recombinant molecule.

In a preferred embodiment, the high mannose protein of interest producedby the host cell of the invention may be a high mannose glycoproteinhaving exposed mannose terminal residues.

Such high mannose protein may be according to another preferredembodiment, a lysosomal enzyme selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseand sialidase. In a preferred embodiment, the lysosomal enzyme may bethe human glucocerebrosidase (GCD). Hereinafter recombinant GCD, rGCD,rhGCD all refer to various forms of recombinant human GCD unlessotherwise indicated.

As previously described, Gaucher's disease, the most prevalent lysosomalstorage disorder, is caused by point mutations in the hGCD (humanglucocerebrosidase) gene (GBA), which result in accumulation of GlcCerin the lysosomes of macrophages. The identification of GCD deficiency asthe primary cause of Gaucher's disease led to the development of enzymereplacement therapy as a therapeutic strategy for this disorder.However, glycosylation plays a crucial role in hGCD activity and uptaketo target cells.

Therefore, according to other preferred embodiments of the presentinvention, suitably glycosylated hGCD is preferably provided bycontrolling the expression of hGCD in plant cell culture, optionally andmore preferably by providing an ER signal and/or otherwise by optionallyand more preferably blocking transportation to the Golgi.

Optionally and preferably, the hGCD has at least one oligosaccharidechain comprising an exposed mannose residue for the treatment orprevention of Gaucher's disease.

Still further, in a particular embodiment, this preferred host cell istransformed or transfected by a recombinant nucleic acid molecule whichfurther comprises an ³⁵S promoter from Cauliflower Mosaic Virus, anoctopine synthase terminator of Agrobacterium tumefaciens and TMV(Tobacco Mosaic Virus) omega translational enhancer element. Accordingto a preferred embodiment, this recombinant nucleic acid moleculecomprises the nucleic acid sequence substantially as denoted by SEQ IDNO: 13 and encodes a high mannose GCD having the amino acid sequencesubstantially as denoted by SEQ ID NOs: 14 or 15.

It should be appreciated that the present invention further provides foran expression vector comprising a nucleic acid molecule encoding abiologically active lysosomal enzyme.

In one preferred embodiment, the expression vector of the inventioncomprises a nucleic acid molecule encoding a biologically active highmannose human glucocerebrosidase (GCD). Preferably, this preferredexpression vector comprises a nucleic recombinant nucleic acid moleculewhich having the nucleic acid sequence substantially as denoted by SEQID NO: 13.

In a second aspect, the present invention relates to a recombinant highmannose protein produced by the host cell of the invention.

In a preferred embodiment, this high mannose protein may be abiologically active high mannose lysosomal enzyme selected from thegroup consisting of glucocerebrosidase (GCD), acid sphingomyelinase,hexosaminidase, α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase and sialidase. Most preferably, this lysosomalenzyme may be human glucocerebrosidase (GCD).

Still further, the invention provides for a recombinant biologicallyactive high mannose lysosomal enzyme having at least one oligosaccharidechain comprising an exposed mannose residue.

According to a preferred embodiment, the recombinant lysosomal enzyme ofthe invention can bind to a mannose receptor on a target cell in atarget site. Preferably, this site may be within a subject sufferingfrom a lysosomal storage disease.

It should be noted that the recombinant lysosomal enzyme has increasedaffinity for the target cell, in comparison with the correspondingaffinity of a naturally occurring lysosomal enzyme for the target cell.In a specific embodiment, the target cell at the target site may be aKupffer cell in the liver of the subject.

In a preferred embodiment, the recombinant lysosomal enzyme may beselected from the group consisting of glucocerebrosidase (GCD), acidsphingomyelinase, hexosaminidase, α-N-acetylgalactosaminidise, acidlipase, α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase.

Most preferably, this recombinant lysosomal enzyme is glucocerebrosidase(GCD).

In a third aspect, the invention relates to a method of producing a highmannose protein. Accordingly, the method of the invention comprises thesteps of: (a) preparing a culture of recombinant host cells transformedor transfected with a recombinant nucleic acid molecules encoding arecombinant protein of interest or with an expression vector comprisingthe recombinant nucleic acid molecules; (b) culturing these host cellculture prepared by step (a) under conditions permitting the expressionof the protein, wherein the host cells produce the protein in a highlymannosylated form; (c) recovering the protein from the cells andharvesting the cells from the culture provided in (a); and (d) purifyingthe protein of step (c) by a suitable protein purification method.

According to a preferred embodiment, the host cell used by this methodis the host cell of the invention.

In another preferred embodiment, the high mannose protein produced bythe method of the invention may be a biologically active high mannoselysosomal enzyme having at least one oligosaccharide chain comprising anexposed mannose residue.

This recombinant enzyme can bind to a mannose receptor on a target cellin a target site. More particularly, the recombinant enzyme produced bythe method of the invention has increased affinity for the target cell,in comparison with the corresponding affinity of a naturally occurringlysosomal enzyme to the target cell. Accordingly, the target cell at thetarget site may be Kupffer cell in the liver of the subject.

In a specific embodiment, this lysosomal enzyme may be selected from thegroup consisting of glucocerebrosidase (GCD), acid sphingomyelinase,hexosaminidase, α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase and sialidase. Most preferably, this lysosomalenzyme may be glucocerebrosidase (GCD).

In another preferred embodiment, the host cell used by the method of theinvention may be a plant root cell selected from the group consisting ofAgrobacterium rihzogenes transformed root cell, celery cell, gingercell, horseradish cell and carrot cell. Most preferably, the plant rootcell is a carrot cell. It should be particularly noted that in themethod of the invention, the transformed host carrot cells are grown insuspension.

In a further aspect, the present invention relates to a method fortreating a subject having lysosomal storage disease using exogenousrecombinant lysosomal enzyme, comprising: (a) providing a recombinantbiologically active form of lysosomal enzyme purified from transformedplant root cells, and capable of efficiently targeting cells abnormallydeficient in the lysosomal enzyme. This recombinant biologically activeenzyme has exposed terminal mannose residues on appendedoligosaccharides; and (b) administering a therapeutically effectiveamount of the recombinant biologically active lysosomal enzyme to thesubject. In a preferred embodiment, the recombinant high mannoselysosomal enzyme used by the method of the invention may be produced bythe host cell of the invention. Preferably, this host cell is a carrotcell.

In another preferred embodiment, the lysosomal enzyme used by the methodof the invention may be a high mannose enzyme comprising at least oneoligosaccharide chain having an exposed mannose residue. Thisrecombinant enzyme can bind to a mannose receptor on a target cell in atarget site within a subject. More preferably, this recombinantlysosomal enzyme has increased affinity for these target cells, incomparison with the corresponding affinity of a naturally occurringlysosomal enzyme to the target cell.

More specifically, the lysosomal enzyme used by the method of theinvention may be selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. Preferably, this lysosomal enzyme is glucocerebrosidase(GCD).

According to a preferred embodiment, the method of the invention istherefore intended for the treatment of a lysosomal storage disease,particularly Gaucher's disease.

In such case the target cell at the target site may be a Kupffer cell inthe liver of the subject.

The invention further provides for a pharmaceutical composition for thetreatment of a lysosomal storage disease comprising as an activeingredient a recombinant biologically active high mannose lysosomalenzyme as defined by the invention. The composition of the invention mayoptionally further comprise pharmaceutically acceptable dilluent,carrier or excipient.

In a specific embodiment, the composition of the invention is intendedfor the treatment of Gaucher's disease. Such composition may preferablycomprise as an effective ingredient a biologically active high mannosehuman glucocerebrosidase (GCD), as defined by the invention.

The invention further relates to the use of a recombinant biologicallyactive high mannose lysosomal enzyme of the invention in the manufactureof a medicament for the treatment or prevention of a lysosomal storagedisease. More particularly, such disease may be Gaucher's disease.

Accordingly, this biologically active lysososomal enzyme is abiologically active high mannose human glucocerebrosidase (GCD), asdefined by the invention.

According to the present invention, there is provided a host cellproducing a high mannose recombinant protein, comprising apolynucleotide encoding the recombinant protein and a signal for causingthe recombinant protein to be produced as a high mannose protein.Preferably, the polynucleotide comprises a first nucleic acid sequenceencoding the protein of interest operably linked to a second nucleicacid sequence encoding a signal peptide. Optionally, the signal peptidecomprises an ER (endoplasmic reticulum) targeting signal peptide.Preferably, the polynucleotide further comprises a third nucleic acidsequence for encoding a vacuolar targeting signal peptide.

Preferably, the signal causes the recombinant protein to be targeted tothe ER. More preferably, the signal comprises a signal peptide forcausing the recombinant protein to be targeted to the ER. Mostpreferably, the polynucleotide comprises a nucleic acid segment forencoding the signal peptide.

Optionally and preferably, the signal causes the recombinant protein toby-pass the Golgi. Preferably, the signal comprises a signal peptide forcausing the recombinant protein to not be targeted to the Golgi. Morepreferably, the polynucleotide comprises a nucleic acid segment forencoding the signal peptide.

Optionally and preferably, the host cell is any one of a eukaryotic anda prokaryotic cell. Optionally, the prokaryotic cell is a bacterialcell, preferably an Agrobacterium tumefaciens cell. Preferably, theeukaryotic cell is a plant cell. More preferably, the plant cell is aplant root cell selected from the group consisting of Agrobacteriumrihzogenes transformed root cell, celery cell, ginger cell, horseradishcell and carrot cell. Most preferably, the plant root cell is a carrotcell.

Preferably, the recombinant polynucleotide comprises a first nucleicacid sequence encoding the protein of interest that is in operable linkwith a second nucleic acid sequence encoding a vacuolar targeting signalpeptide derived from the basic tobacco chitinase A gene, which vacuolarsignal peptide has the amino acid sequence as denoted by SEQ ID NO: 2,wherein the first nucleic acid sequence is optionally further operablylinked to a third nucleic acid sequence encoding an ER (endoplasmicreticulum) targeting signal peptide as denoted by SEQ ID NO: 1.

More preferably, the recombinant polynucleotide further comprises apromoter that is functional in plant cells, wherein the promoter isoperably linked to the recombinant molecule.

Most preferably, the recombinant polynucleotide further comprises aterminator that is functional in plant cells, wherein the terminator isoperably linked to the recombinant molecule.

Also most preferably, the recombinant polynucleotide optionally furthercomprises additional control, promoting and regulatory elements and/orselectable markers, wherein the regulatory elements are operably linkedto the recombinant molecule.

Preferably, the high mannose protein is a high mannose glycoproteinhaving glycosylation with at least one exposed mannose residue. Morepreferably, the high mannose protein is a biologically active highmannose lysosomal enzyme selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseand sialidase

Most preferably, the lysosomal enzyme is human glucocerebrosidase (GCD).

Preferably, the GCD comprises the amino acid sequence substantially asdenoted by SEQ ID NO: 8, encoded by the nucleic acid sequence as denotedby SEQ ID NO: 7.

More preferably, the cell is transformed or transfected with arecombinant polynucleotide or with an expression vector comprising themolecule, which recombinant polynucleotide further comprises an ³⁵Spromoter from Cauliflower Mosaic Virus, an octopine synthase terminatorof Agrobacterium tumefaciens, and the regulatory element is the TMV(Tobacco Mosaic Virus) omega translational enhancer element, and havingthe nucleic acid sequence substantially as denoted by SEQ ID NO: 13encoding GCD having the amino acid sequence substantially as denoted bySEQ ID NOs: 14 or 15.

According to preferred embodiments, there is provided a recombinant highmannose protein produced by the host cell described above.

Preferably, the high mannose protein is a biologically active highmannose lysosomal enzyme selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseand sialidase.

More preferably, the lysosomal enzyme is human glucocerebrosidase (GCD).

According to other preferred embodiments of the present invention, thereis provided a recombinant biologically active high mannose lysosomalenzyme having at least one oligosaccharide chain comprising an exposedmannose residue.

According to still other preferred embodiments, there is provided arecombinant protein, comprising a first portion having signal peptideactivity and a second portion having lysosomal enzyme activity, thefirst portion causing the second portion to be processed in a plant cellwith at least one oligosaccharide chain comprising an exposed mannoseresidue.

Preferably, the lysosomal enzyme comprises a protein for the treatmentor prevention of Gaucher's disease.

More preferably, the protein comprises hGCD.

Preferably, the first portion comprises a plant cell ER targeting signalpeptide. More preferably, the recombinant enzyme can bind to a mannosereceptor on a target cell in a target site within a subject sufferingfrom a lysosomal storage disease. Most preferably, the recombinantlysosomal enzyme has increased affinity for the target cell, incomparison with the corresponding affinity of a naturally occurringlysosomal enzyme for the target cell.

Also most preferably, the recombinant lysosomal enzyme is selected fromthe group consisting of glucocerebrosidase (GCD), acid sphingomyelinase,hexosaminidase, α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase.

Preferably, the recombinant lysosomal enzyme is glucocerebrosidase(GCD).

Also preferably, the target cell at the target site is a Kupffer cell inthe liver of the subject.

According to still other preferred embodiments there is provided arecombinant high mannose protein, produced in plant cell culture.Preferably, the protein features a plant signal peptide for targeting aprotein to the ER.

More preferably, the plant signal peptide comprises a peptide fortargeting the protein to the ER in a root plant cell culture. Mostpreferably, the root plant cell culture comprises carrot cells.

According to yet other preferred embodiments there is provided arecombinant high mannose hGCD protein, produced in plant cell culture.

According to still other preferred embodiments there is provided use ofa plant cell culture for producing a high mannose protein.

According to other preferred embodiments there is provided a method ofproducing a high mannose protein comprising: preparing a culture ofrecombinant host cells transformed or transfected with a recombinantpolynucleotide encoding for a recombinant protein; culturing the hostcell culture under conditions permitting the expression of the protein,wherein the host cells produce the protein in a highly mannosylatedform.

Preferably, the host cell culture is cultured in suspension. Morepreferably, the method further comprises purifying the protein.

According to other preferred embodiments, the method is performed withthe host cell as previously described. Preferably, the high mannoseprotein is a biologically active high mannose lysosomal enzyme having atleast one oligosaccharide chain comprising an exposed mannose residue.More preferably, the recombinant enzyme binds to a mannose receptor on atarget cell in a target site. Most preferably, the recombinant enzymehas increased affinity for the target cell, in comparison with thecorresponding affinity of a naturally occurring lysosomal enzyme to thetarget cell.

Preferably, the lysosomal enzyme is selected from the group consistingof glucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseand sialidase.

More preferably, the lysosomal enzyme is glucocerebrosidase (GCD). Mostpreferably, the target cell at the target site is Kupffer cell in theliver of the subject.

Preferably, the host cell is a plant root cell selected from the groupconsisting of Agrobacterium rihzogenes transformed root cell, celerycell, ginger cell, horseradish cell and carrot cell.

More preferably, the plant root cell is a carrot cell.

Most preferably, the transformed host carrot cells are grown insuspension.

According to still other preferred embodiments there is provided amethod for treating a subject having lysosomal storage disease usingexogenous recombinant lysosomal enzyme, comprising: providing arecombinant biologically active form of lysosomal enzyme purified fromtransformed plant root cells, and capable of efficiently targeting cellsabnormally deficient in the lysosomal enzyme, wherein the recombinantbiologically active enzyme has exposed terminal mannose residues onappended oligosaccharides; and administering a therapeutically effectiveamount of the recombinant biologically active lysosomal enzyme to thesubject. This method may optionally be performed with any host celland/or protein as previous described.

Preferably, the recombinant enzyme can bind to a mannose receptor on atarget cell in a target site within a subject. More preferably, therecombinant lysosomal enzyme has increased affinity for the target cell,in comparison with the corresponding affinity of a naturally occurringlysosomal enzyme to the target cell. Most preferably, the lysosomalenzyme is selected from the group consisting of glucocerebrosidase(GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. Also most preferably, the lysosomal enzyme isglucocerebrosidase (GCD).

Also most preferably, the lysosomal storage disease is Gaucher'sdisease. Also most preferably, the target cell at the target site is aKupffer cell in the liver of the subject.

According to still other preferred embodiments there is provided apharmaceutical composition for the treatment of a lysosomal storagedisease comprising as an active ingredient a recombinant biologicallyactive high mannose lysosomal enzyme as described above, whichcomposition optionally further comprises pharmaceutically acceptabledilluent, carrier or excipient. Preferably, the lysosomal storagedisease is Gaucher's disease. More preferably, the recombinant lysosomalenzyme is a biologically active high mannose human glucocerebrosidase(GCD).

According to still other preferred embodiments there is provided the useof a recombinant biologically active high mannose lysosomal enzyme asdescribed above, in the manufacture of a medicament for the treatment orprevention of a lysosomal storage disease. Preferably, the disease isGaucher's disease. More preferably, the biologically active lysososomalenzyme is a biologically active high mannose human glucocerebrosidase(GCD).

The invention will be further described on the hand of the followingfigures, which are illustrative only and do not limit the scope of theinvention which is also defined by the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

The invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

FIG. 1A-1B

1A shows the resulting expression cassette comprising ³⁵S promoter fromCauliflower Mosaic Virus, TMV (Tobacco Mosaic Virus) omega translationalenhancer element, ER targeting signal, the human GCD sequence (alsodenoted by SEQ ID NO: 7), vacuolar signal and octopine synthaseterminator sequence from Agrobacterium tumefaciens.

1B shows a schematic map of pGreenII plasmid backbone.

FIG. 2 shows Western blot analysis of hGCD transformed cell extractsusing anti hGCD specific antibody. Standard Cerezyme (lane 1) was usedas a positive control, untransformed callus was used as negative control(lane 2), various selected calli extracts are shown in lanes 3-8.

FIG. 3A-3C shows the first step of purification of rhGCD on a strongcation exchange resin (Macro-Prep high-S support, Bio-Rad), packed in aXK column (2.6×20 cm). The column was integrated with an AKTA primesystem (Amersham Pharmacia Biotech) that allows conductivity monitoring,pH and absorbency at 280 nm. Elution of the rh-GCD was obtained withequilibration buffer containing 600 mM NaCl.

FIG. 3A represents a standard run of this purification step. Thefractions collected during the run were monitored by enzyme activityassay, as shown by FIG. 3B, and tubes exhibiting enzymatic activity (inthe elution peak) were pooled. FIG. 3C shows coomassie-blue stain ofelution fractions assayed for activity.

FIGS. 3D-3F show corresponding graphs as for FIGS. 3A-3C but for thesecond column.

FIG. 4A-C: shows the final purification step of the recombinant hGCD ona hydrophobic interaction resin (TSK gel, Toyopearl Phenyl-650C, TosohCorp.), packed in a XK column (2.6×20 cm). The column was integratedwith an AKTA prime system (Amersham Pharmacia Biotech) that allowsconductivity monitoring, pH and absorbency at 280 nm. The GCD elutionpool from the previous column was loaded at 6 ml/min followed by washingwith equilibration buffer until the UV absorbance reach the baseline.The pure GCD was eluted by 10 mM citric buffer containing 50% ethanol.

FIG. 4A represents a standard run of this purification step.

FIG. 4B shows the fractions collected during the run that were monitoredby enzyme activity assay.

FIG. 4C shows coomassie-blue stain of elution fractions assayed foractivity.

FIG. 5 shows activity of recombinant hGCD following uptake by peritonealmacrophages (FIGS. 5A-5C), while FIG. 5D shows a Western blot ofrecombinant GCD according to the present invention.

FIG. 6 shows comparative glycosylation structures for rGCD according tothe present invention and that of Cerezyme™.

FIG. 7 shows glycosylation structures for rGCD according to the presentinvention.

FIG. 8 a-8 d shows additional N-glycan glycosylation structures for rGCDaccording to the present invention.

FIGS. 9 a-9 b show the antigenic and electrophoretic identity ofpurified recombinant human GCD of the present invention and a commercialhuman GCD (Cerezyme®) recombinantly produced in mammalian CHO cells.FIG. 9 a is a Coomassie blue stained SDS-PAGE analysis of the plantproduced hGCD of the invention (lanes 1 and 2, 5 and 10 μg of protein,respectively) and Cerezyme®, (lanes 3 and 4, 5 and 10 μg protein,respectively). FIG. 9 b is a Western blot analysis of SDS-PAGE separatedrecombinant human GCD (lanes 1 and 2, 50 and 10 ng respectively) of thepresent invention compared to the commercial Cerezyme® enzyme.SDS-PAGE-separated proteins were blotted onto nitrocellulose (lanes 3and 4, 50 and 100 ng antigen, respectively), and immunodetected using apolyclonal anti-GCD antibody and peroxidase-conjugated goat anti-rabbitHRP secondary antibody. Note the consistency of size and immunereactivity between the plant recombinant GCD of the present inventionand the mammalian-cell (CHO) prepared enzyme (Cerezyme®). MW=molecularweight standard markers;

FIGS. 10 a-10 b are schematic representations of the glycan structuresof the recombinant human GCD of the present invention. FIG. 10 a showsthe results of a major glycan structure analysis of the GCD, indicatingall structures and their relative amounts based on HPLC, enzyme arraydigests and MALDI. Retention time of individual glycans is compared tothe retention times of a standard partial hydrolysis of dextran giving aladder of glucose units (GU). FIG. 10 b shows the glycan structures ofthe mammalian-cell (CHO) prepared enzyme (Cerezyme®), before and afterthe in-vitro modification process. Note the predominance of the xyloseand exposed mannose glycosides in the recombinant human GCD of thepresent invention;

FIG. 11 is a HP-anion exchange chromatography analysis of the gycanprofile of the recombinant human GCD of the present invention, showingthe consistent and reproducible glycan structure of recombinant humanGCD from batch to batch;

FIG. 12 is a kinetic analysis showing the identical catalytic kineticscharacteristic of both recombinant human GCD of the invention (opentriangles) and the mammalian-cell (CHO) prepared enzyme (Cerezyme®)(closed squares). Recombinant human GCD of the invention and Cerezyme®(0.2 μg) were assayed using C6-NBDGlcCer (5 min, 37° C.) in MES buffer(50 mM, pH 5.5). Michaelis-Menten kinetics was analyzed using GraphPadPrism software. Data are means of two independent experiments;

DETAILED DESCRIPTION OF THE INVENTION

Proteins for pharmaceutical use have been traditionally produced inmammalian or bacterial expression systems. In the past few years apromising new expression system was found in plants. Due to the relativesimplicity of introducing new genes and potential for mass production ofproteins and peptides, ‘molecular pharming’ is becoming increasinglypopular as a protein expression system.

One of the major differences between mammalian and plant proteinexpression system is the variation of protein glycosylation sequences,caused by the differences in biosynthetic pathways. Glycosylation wasshown to have a profound effect on activity, folding, stability,solubility, susceptibility to proteases, blood clearance rate andantigenic potential of proteins. Hence, any protein production in plantsshould take into consideration the potential ramifications of plantglycosylation.

This is well illustrated by the difficulties encountered in previousattempts to produce biologically active mammalian proteins in plants.For example, U.S. Pat. No. 5,929,304, to Radin et al (Crop Tech, Inc)discloses the production, in tobacco plants, of a human α-L-iduronase(IDUA) and a glucocerebrosidase (hGC), by insertion of the relevanthuman lysosomal enzyme coding sequences into an expression cassette forbinary plasmid for A. tumefaciens-mediated transformation of tobaccoplants. Despite demonstration of recombinant human lysosomal proteinproduction in the transgenic plants, and the detection of catalyticactivity in the recombinant protein, no binding to or uptake into targetcells was disclosed, and the lysosomal enzyme compositions remainedunsuitable for therapeutic applications, presumably due to the absenceof accurate glycosylation of the protein, and subsequent inability ofthe polypeptides to interact efficiently with their target cells/tissuethough a specific receptor.

Carbohydrate moiety is one of the most common post-translationalmodifications of proteins. Protein glycosylation is divided into twocategories: N-linked and O-linked. The two types differ in amino acid towhich the glycan moiety is attached on protein—N-linked are attached toAsn residues, while O-linked are attached to Ser or Thr residues. Inaddition, the glycan sequences of each type bears unique distinguishingfeatures. Of the two types, N-linked glycosylation is the more abundant,and its effect on proteins has been extensively studied. O-linkedglycans, on other hand are relatively scarce, and less information isavailable regarding their influence on proteins. The majority of dataavailable on protein glycosylation in plants focuses on N-linked, ratherthan O-linked glycans.

The present invention describes herein a plant expression system basedon transgenic plant cells, which are preferably root cells, optionallyand preferably grown in suspension. This expression system isparticularly designed for efficient production of a high mannose proteinof interest. The term “high mannose” includes glycosylation having atleast one exposed mannose residue.

Thus, in a first aspect, the present invention relates to a host cellproducing a high mannose recombinant protein of interest. Preferably,the recombinant protein features an ER (endoplasmic reticulum) signalpeptide, more preferably an ER targeting signal peptide. Alternativelyor additionally, the recombinant protein features a signal that causesthe protein to by-pass the Golgi. The signal preferably enables therecombinant protein to feature high mannose glycosylation, morepreferably by retaining such glycosylation, and most preferably bytargeting the ER and/or by-passing the Golgi. As described in greaterdetail herein, such a signal is preferably implemented as a signalpeptide, which more preferably forms part of the protein sequence,optionally and more preferably through engineering the protein to alsofeature the signal peptide as part of the protein. It should be notedthat the signal may optionally be a targeting signal, a retentionsignal, an avoidance (by-pass) signal, or any combination thereof, orany other type of signal capable of providing the desired high mannoseglycosylation structure.

Without wishing to be limited by a single hypothesis, it would appearthat the use of an ER targeting signal with the recombinant proteinbeing produced in plant cell culture was able to overcome transportationto the Golgi, and hence to retain the desired high mannoseglycosylation. Optionally, any type of mechanism which is capable toproduce high mannose glycosylation, including any type of mechanism toby-pass the Golgi, may be used in accordance with the present invention.ER targeting signal peptides are well known in the art; they areN-terminal signal peptides. Optionally any suitable ER targeting signalpeptide may be used with the present invention.

A host cell according to the present invention may optionally betransformed or transfected (permanently and/or transiently) with arecombinant nucleic acid molecule encoding a protein of interest or withan expression vector comprising the nucleic acid molecule. Such nucleicacid molecule comprises a first nucleic acid sequence encoding theprotein of interest, optionally and preferably operably linked to asecond nucleic acid sequence encoding a vacuolar targeting signalpeptide. It should be noted that as used herein, the term “operably”linked does not necessarily refer to physical linkage. The first nucleicacid sequence may optionally and preferably further be operably linkedto a third nucleic acid sequence encoding an ER (endoplasmic reticulum)targeting signal peptide. In one embodiment, the cell of the inventionis characterized in that the protein of interest is produced by the cellin a form that includes at least one exposed mannose residue, but ispreferably a highly mannosylated form. In a more preferred embodiment,the cell of the protein of interest is produced by the cell in a formthat includes an exposed mannose and at least one xylose residue, in yeta more preferred embodiment, in a form that further includes an exposedmannose and at least one fucose residue. In a most preferred embodiment,the protein is produced by the cell in a form that includes an exposedmannose, a core α (1,2) xylose residue and a core α-(1,3) fucoseresidue.

“Cells”, “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cells but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggeneration due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. “Cell” or “hostcell” as used herein refers to cells which can be transformed with nakedDNA or expression vectors constructed using recombinant DNA techniques.As used herein, the term “transfection” means the introduction of anucleic acid, e.g., naked DNA or an expression vector, into a recipientcells by nucleic acid-mediated gene transfer. “Transformation”, as usedherein, refers to a process in which a cell's genotype is changed as aresult of the cellular uptake of exogenous DNA or RNA, and, for example,the transformed cell expresses a recombinant form of the desiredprotein.

It should be appreciated that a drug resistance or other selectablemarker is intended in part to facilitate the selection of thetransformants. Additionally, the presence of a selectable marker, suchas drug resistance marker may be of use in keeping contaminatingmicroorganisms from multiplying in the culture medium. Such a pureculture of the transformed host cell would be obtained by culturing thecells under conditions which are required for the induced phenotype'ssurvival.

As indicated above, the host cells of the invention may be transfectedor transformed with a nucleic acid molecule. As used herein, the term“nucleic acid” refers to polynucleotides such as deoxyribonucleic acid(DNA), and, where appropriate, ribonucleic acid (RNA). The terms shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides.

In yet another embodiment, the cell of the invention may be transfectedor transformed with an expression vector comprising the recombinantnucleic acid molecule. “Expression Vectors”, as used herein, encompassvectors such as plasmids, viruses, bacteriophage, integratable DNAfragments, and other vehicles, which enable the integration of DNAfragments into the genome of the host. Expression vectors are typicallyself-replicating DNA or RNA constructs containing the desired gene orits fragments, and operably linked genetic control elements that arerecognized in a suitable host cell and effect expression of the desiredgenes. These control elements are capable of effecting expression withina suitable host. Generally, the genetic control elements can include aprokaryotic promoter system or a eukaryotic promoter expression controlsystem. Such system typically includes a transcriptional promoter, anoptional operator to control the onset of transcription, transcriptionenhancers to elevate the level of RNA expression, a sequence thatencodes a suitable ribosome binding site, RNA splice junctions,sequences that terminate transcription and translation and so forth.Expression vectors usually contain an origin of replication that allowsthe vector to replicate independently of the host cell.

Plasmids are the most commonly used form of vector but other forms ofvectors which serves an equivalent function and which are, or become,known in the art are suitable for use herein. See, e.g., Pouwels et al.Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier,N.Y.; and Rodriquez, et al. (eds.) Vectors: a Survey of MolecularCloning Vectors and their Uses, Buttersworth, Boston, Mass. (1988),which are incorporated herein by reference.

In general, such vectors contain, in addition, specific genes which arecapable of providing phenotypic selection in transformed cells. The useof prokaryotic and eukaryotic viral expression vectors to express thegenes coding for the polypeptides of the present invention are alsocontemplated.

Optionally, the vector may be a general plant vector (as described withregard to the Examples below). Alternatively, the vector may optionallybe specific for root cells.

In one preferred embodiment, the cell of the invention may be aeukaryotic or prokaryotic cell.

In a specific embodiment, the cell of the invention is a prokaryoticcell, preferably, a bacterial cell, most preferably, an Agrobacteriumtumefaciens cell. These cells are used for infecting the preferred planthost cells described below.

In another preferred embodiment, the cell of the invention may be aeukaryotic cell, preferably, a plant cell, and most preferably, a plantroot cell selected from the group consisting of Agrobacterium rihzogenestransformed plant root cell, celery cell, ginger cell, horseradish celland carrot cell.

In a preferred embodiment, the plant root cell is a carrot cell. Itshould be noted that the transformed carrot cells of the invention aregrown in suspension. As mentioned above and described in the Examples,these cells were transformed with the Agrobacterium tumefaciens cells ofthe invention.

The expression vectors or recombinant nucleic acid molecules used fortransfecting or transforming the host cells of the invention may befurther modified according to methods known to those skilled in the artto add, remove, or otherwise modify peptide signal sequences to altersignal peptide cleavage or to increase or change the targeting of theexpressed lysosomal enzyme through the plant endomembrane system. Forexample, but not by way of limitation, the expression construct can bespecifically engineered to target the lysosomal enzyme for secretion, orvacuolar localization, or retention in the endoplasmic reticulum (ER).

In one embodiment, the expression vector or recombinant nucleic acidmolecule, can be engineered to incorporate a nucleotide sequence thatencodes a signal targeting the lysosomal enzyme to the plant vacuole.For example, and not by way of limitation, the recombinant nucleic acidmolecule comprised within the host cell of the invention, comprises afirst nucleic acid sequence encoding a lysosomal enzyme that is inoperable linkage with a second nucleic acid sequence encoding a vacuolartargeting signal peptide derived from the basic tobacco chitinase Agene. This vacuolar signal peptide has the amino acid sequence asdenoted by SEQ ID NO: 2. The first nucleic acid sequence may beoptionally further linked in an operable linkage with a third nucleicacid sequence encoding an ER (endoplasmic reticulum) targeting signalpeptide as denoted by SEQ ID NO: 1. In one embodiment, the recombinantnucleic acid molecule comprised within the host cell of the inventionfurther comprises a promoter that is functional in plant cells. Thispromoter should be operably linked to the recombinant molecule of theinvention.

The term “operably linked” is used herein for indicating that a firstnucleic acid sequence is operably linked with a second nucleic acidsequence when the first nucleic acid sequence is placed in a functionalrelationship with the second nucleic acid sequence. For instance, apromoter is operably linked to a coding sequence if the promoter affectsthe transcription or expression of the coding sequence. Optionally andpreferably, operably linked DNA sequences are contiguous (e.g.physically linked) and, where necessary to join two protein-codingregions, in the same reading frame. Thus, a DNA sequence and aregulatory sequence(s) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

In another embodiment, this recombinant nucleic acid molecule mayoptionally further comprise an operably linked terminator which ispreferably functional in plant cells. The recombinant nucleic acidmolecule of the invention may optionally further comprise additionalcontrol, promoting and regulatory elements and/or selectable markers. Itshould be noted that these regulatory elements are operably linked tothe recombinant molecule.

Regulatory elements that may be used in the expression constructsinclude promoters which may be either heterologous or homologous to theplant cell. The promoter may be a plant promoter or a non-plant promoterwhich is capable of driving high levels transcription of a linkedsequence in plant cells and plants. Non-limiting examples of plantpromoters that may be used effectively in practicing the inventioninclude cauliflower mosaic virus (CaMV) ³⁵S, rbcS, the promoter for thechlorophyll a/b binding protein, AdhI, NOS and HMG2, or modifications orderivatives thereof. The promoter may be either constitutive orinducible. For example, and not by way of limitation, an induciblepromoter can be a promoter that promotes expression or increasedexpression of the lysosomal enzyme nucleotide sequence after mechanicalgene activation (MGA) of the plant, plant tissue or plant cell.

The expression vectors used for transfecting or transforming the hostcells of the invention can be additionally modified according to methodsknown to those skilled in the art to enhance or optimize heterologousgene expression in plants and plant cells. Such modifications includebut are not limited to mutating DNA regulatory elements to increasepromoter strength or to alter the protein of interest.

In a preferred embodiment, the high mannose protein of interest producedby the host cell of the invention may be a high mannose glycoproteinhaving at least one exposed mannose residue (at least one terminalmannose residue).

Such high mannose protein may be according to another preferredembodiment, a lysosomal enzyme selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseand sialidase

The term “lysosomal enzyme”, as used herein with respect to any suchenzyme and product produced in a plant expression system described bythe invention, refers to a recombinant peptide expressed in a transgenicplant cell from a nucleotide sequence encoding a human or animallysosomal enzyme, a modified human or animal lysosomal enzyme, or afragment, derivative or modification of such enzyme. Useful modifiedhuman or animal lysosomal enzymes include but are not limited to humanor animal lysosomal enzymes having one or several naturally occurring orartificially introduced amino acid additions, deletions and/orsubstitutions.

Soluble lysosomal enzymes share initial steps of biosynthesis withsecretory proteins, i.e., synthesis on the ribosome, binding of theN-terminal signal peptide to the surface of the rough endoplasmicreticulum (ER), transport into the lumen of the ER where the signalpeptide is cleaved, and addition of oligosaccharides to specificasparagine residues (N-linked), followed by further modifications of thenascent protein in the Golgi apparatus [von Figura and Hasilik, Annu.Rev. Biochem. 55:167-193 (1986)]. The N-linked oligosaccharides can becomplex, diverse and heterogeneous, and may contain high-mannoseresidues. The proteins undergo further processing in a post-ER,pre-Golgi compartment and in the cis-Golgi to form either an N-linkedmannose 6-phosphate (M-6-P) oligosaccharide-dependent or N-linked M-6-Poligosaccharide-independent recognition signal for lysosomal localizedenzymes [Kornfeld & Mellman, Ann. Rev. Cell Biol., 5:483-525 (1989);Kaplan et al., Proc. Natl. Acad. Sci. USA 74:2026 (1977)]. The presenceof the M-6-P recognition signal results in the binding of the enzyme toM-6-P receptors (MPR). These bound enzymes remain in the cell, areeventually packaged into lysosomes, and are thus segregated fromproteins targeted for secretion or to the plasma membrane.

In a preferred embodiment, the lysosomal enzyme may be the humanglucocerebrosidase (GCD).

Still further, in a particular embodiment, this preferred host cell istransformed or transfected by a recombinant nucleic acid molecule whichfurther comprises an ³⁵S promoter from Cauliflower Mosaic Virus,preferably, having the nucleic acid sequence as denoted by SEQ ID NO: 9,an octopine synthase terminator of Agrobacterium tumefaciens,preferably, having the nucleic acid sequence as denoted by SEQ ID NO: 12and TMV (Tobacco Mosaic Virus) omega translational enhancer element.According to a preferred embodiment, this recombinant nucleic acidmolecule comprises the nucleic acid sequence substantially as denoted bySEQ ID NO: 13 and encodes a high mannose GCD having the amino acidsequence substantially as denoted by SEQ ID NOs: 14 or 15.

It should be appreciated that the present invention further provides foran expression vector comprising a nucleic acid molecule encoding abiologically active high mannose lysosomal enzyme.

In one preferred embodiment of the aspect, the expression vector of theinvention comprises a nucleic acid molecule encoding a biologicallyactive high mannose human glucocerebrosidase (GCD). Preferably, thispreferred expression vector comprises a recombinant nucleic acidmolecule which having the nucleic acid sequence substantially as denotedby SEQ ID NO: 13. According to a specific embodiment, a preferredexpression vector utilizes the pGREEN II plasmid as described by thefollowing Example 1.

It should be further noted, that the invention provides for anexpression cassette comprised within the expression vector describedabove.

In a second aspect, the present invention relates to a recombinant highmannose protein produced by the host cell of the invention.

In a preferred embodiment, this high mannose protein may be abiologically active high mannose lysosomal enzyme selected from thegroup consisting of glucocerebrosidase (GCD), acid sphingomyelinase,hexosaminidase, α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase and sialidase. Most preferably, this lysosomalenzyme may be human glucocerebrosidase (GCD).

The term “biologically active” is used herein with respect to anyrecombinant lysosomal enzyme produced in a plant expression system tomean that the recombinant lysosomal enzyme is able to hydrolyze eitherthe natural substrate, or an analogue or synthetic substrate of thecorresponding human or animal lysosomal enzyme, at detectable levels.

Still further, the invention provides for a recombinant biologicallyactive high mannose lysosomal enzyme having at least one oligosaccharidechain comprising an exposed mannose residue.

According to a preferred embodiment, the recombinant lysosomal enzyme ofthe invention can bind to a mannose receptor on a target cell in atarget site. Preferably, this site may be within a subject sufferingfrom a lysosomal storage disease.

Optionally and more preferably, the recombinant lysosomal enzyme hasincreased affinity for the target cell, in comparison with thecorresponding affinity of a naturally occurring lysosomal enzyme for thetarget cell. In a specific embodiment, the target cell at the targetsite may be a Kupffer cell in the liver of the subject.

In a preferred embodiment, the recombinant lysosomal enzyme may beselected from the group consisting of glucocerebrosidase (GCD), acidsphingomyelinase, hexosaminidase, α-N-acetylgalactosaminidise, acidlipase, α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase.

Most preferably, this recombinant lysosomal enzyme is glucocerebrosidase(GCD).

In a third aspect, the invention relates to a method of producing a highmannose protein. Accordingly, the method of the invention comprises thesteps of: (a) preparing a culture of recombinant host cells transformedor transfected with a recombinant nucleic acid molecules encoding for arecombinant protein of interest or with an expression vector comprisingthe recombinant nucleic acid molecules; (b) culturing the host cellculture prepared by step (a) in suspension under conditions permittingthe expression of the high mannose protein, wherein the host cellsproduce the protein in a highly mannosylated form; (c) harvesting thecells from the culture provided in (a) and recovering the protein fromthe cells; and (d) purifying the protein of step (c) by a suitableprotein purification method.

Optionally and preferably, the recombinant protein may be produced byplant cells according to the present invention by culturing in a devicedescribed with regard to U.S. Pat. No. 6,391,638, issued on May 21, 2002and hereby incorporated by reference as if fully set forth herein.Conditions for culturing plant cells in suspension with this device aredescribed with regard to the US patent application entitled “CELL/TISSUECULTURING DEVICE, SYSTEM AND METHOD” by one of the present inventors andowned in common with the present application, which is herebyincorporated by reference as if fully set forth herein and which wasfiled on the same day as the present application.

A particular and non limiting example for recovering and purification ofa high mannose protein of interest produced by the method of theinvention may be found in the following Examples. The Examples show thata recombinant h-GCD produced by the invention was unexpectedly bound tointernal membrane of the transformed carrot cells of the invention andnot secreted to the medium. The soluble rh-GCD may be separated fromcell debris and other insoluble component according to means known inthe art such as filtration or precipitation. For Example, following afreeze-thaw cycle, the cells undergo breakage and release ofintracellular soluble proteins, whereas the h-GCD remains bound toinsoluble membrane debris. This soluble and insoluble membrane debrismixture was next centrifuged and the soluble fraction was removed thussimplifying the purification. The membrane bound h-GCD can then bedissolved by mechanical disruption in the presence of a mild detergent,protease inhibitors and neutralizing oxidation reagent. The solubleenzyme may be further purified using chromatography techniques, such ascation exchange and hydrophobic interaction chromatography columns.During rh-GCD production in the bio-reactor and the purification processthe h-GCD identity, yield, purity and enzyme activity can be determinedby one or more biochemical assays. Including but not limited todetecting hydrolysis of the enzyme's substrate or a substrate analogue,SDS-polyacrylamide gel electrophoresis analysis and immunologicalanalyses such as ELISA and Western blot.

According to a preferred embodiment, the host cell used by this methodcomprises the host cell of the invention.

In another preferred embodiment, the high mannose protein produced bythe method of the invention may be a biologically active high mannoselysosomal enzyme having at least one oligosaccharide chain comprising anexposed mannose residue.

This recombinant enzyme can bind to a mannose receptor on a target cellin a target site. More particularly, the recombinant enzyme produced bythe method of the invention has increased affinity for the target cell,in comparison with the corresponding affinity of a naturally occurringlysosomal enzyme to the target cell. Accordingly, the target cell at thetarget site may be Kupffer cell in the liver of the subject.

In a specific embodiment, this lysosomal enzyme may be selected from thegroup consisting of glucocerebrosidase (GCD), acid sphingomyelinase,hexosaminidase, α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase and sialidase. Most preferably, this lysosomalenzyme may be glucocerebrosidase (GCD).

In another preferred embodiment, the host cell used by the method of theinvention may be a plant root cell selected from the group consisting ofAgrobacterium rihzogenes transformed root cell, celery cell, gingercell, horseradish cell and carrot cell. Most preferably, the plant rootcell is a carrot cell. It should be particularly noted that thetransformed host carrot cells are grown in suspension.

In a further aspect, the present invention relates to a method fortreating a subject, preferably a mammalian subject, having lysosomalstorage disease by using exogenous recombinant lysosomal enzyme.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures

Plasmid vectors

-   -   CE-T—Was constructed from plasmid CE obtained from Prof. Galili        [U.S. Pat. No. 5,367,110 Nov. 22, (1994)].

Plasmid CE was digested with SalI.

The SalI cohesive end was made blunt-ended using the large fragment ofDNA polymerase I. Then the plasmid was digested with PstI and ligated toa DNA fragment coding for the ER targeting signal from the basicendochitinase gene [Arabidopsis thaliana]ATGAAGACTAATCTTTTTCTCTTTCTCATCTTTTCA

CTTCTCCTATCATTATCCTCGGCCGAATTC, and vacuolar targeting signal fromTobacco chitinase A: GATCTTTTAGTCGATACTATG digested with SmaI and PstI.

-   -   pGREENII—obtained from Dr. P. Mullineaux [Roger P. Hellens et        al., (2000)

Plant Mol. Bio. 42:819-832]. Expression from the pGREEN II vector iscontrolled by the 35S promoter from Cauliflower Mosaic Virus, the TMV(Tobacco Mosaic Virus) omega translational enhancer element and theoctopine synthase terminator sequence from Agrobacterium tumefaciens.

cDNA

hGCD—obtained from ATCC (Accession No. 65696), GC-2.2 [GCS-2 kb;lambda-EZZ-gamma3 Homo sapiens] containing glucosidase beta acid[glucocerebrosidase]. Insert lengths (kb): 2.20; Tissue: fibroblastWI-38 cell.

Construction of Expression Plasmid

The cDNA coding for hGCD (ATTC clone number 65696) was amplified usingthe forward: 5′ CAGAATTCGCCCGCCCCTGCA 3′ and the reverse: 5′CTCAGATCTTGGCGATGCCACA 3′ primers. The purified PCR DNA product wasdigested with endonucleases EcoRI and BglII (see recognition sequencesunderlined in the primers) and ligated into an intermediate vectorhaving an expression cassette E-T digested with the same enzymes. Theexpression cassette was cut and eluted from the intermediate vector andligated into the binary vector pGREENII using restriction enzymes SmaIand XbaI, forming the final expression vector. Kanamycine resistance isconferred by the NPTII gene driven by the nos promoter obtained togetherwith the pGREEN vector (FIG. 1B). The resulting expression cassette ispresented by FIG. 1A.

The resulting plasmid was sequenced to ensure correct in-frame fusion ofthe signals using the following sequencing primers: 5′ 35S promoter: 5′CTCAGAAGACCAGAGGGC 3′, and the 3′ terminator: 5′CAAAGCGGCCATCGTGC 3′.

Establishment of Carrot Callus and Cell Suspension Cultures

Establishment of carrot callus and cell suspension cultures we preformedas described previously by Torres K. C. (Tissue culture techniques forhorticular crops, p.p. 111, 169).

Transformation of Carrot Cells and Isolation of Transformed Cells.

Transformation of carrot cells was preformed using Agrobacteriumtransformation by an adaptation of a method described previously[Wurtele, E. S. and Bulka, K. Plant Sci. 61:253-262 (1989)]. Cellsgrowing in liquid media were used throughout the process instead ofcalli. Incubation and growth times were adapted for transformation ofcells in liquid culture. Briefly, Agrobacteria were transformed with thepGREEN II vector by electroporation [den Dulk-Ra, A. and Hooykaas, P. J.(1995) Methods Mol. Biol. 55:63-72] and then selected using 30 mg/mlparomomycine antibiotic. Carrot cells were transformed with Agrobacteriaand selected using 60 mg/ml of paromomycine antibiotics in liquid media.

Screening of Transformed Carrot Cells for Isolation of Calli ExpressingHigh Levels of GCD

14 days following transformation, cells from culture were plated onsolid media at dilution of 3% packed cell volume for the formation ofcalli from individual clusters of cells. When individual calli reached1-2 cm in diameter, the cells were homogenized in SDS sample buffer andthe resulting protein extracts were separated on SDS-PAGE [Laemmli U.,(1970) Nature 227:680-685] and transferred to nitrocellulose membrane(hybond C nitrocellulose, 0.45 micron. Catalog No: RPN203C From AmershamLife Science). Western blot for detection of GCD was performed usingpolyclonal anti hGCD antibodies (described herein below). Calliexpressing significant levels of GCD were expanded and transferred togrowth in liquid media for scale up, protein purification and analysis.

Preparation of Polyclonal Antibodies

75 micrograms recombinant GCD (Cerezyme™) were suspended in 3 mlcomplete Freund's adjuvant and injected to each of two rabbits. Eachrabbit was given a booster injection after two weeks. The rabbits werebled about 10 days after the booster injection and again at one weekintervals until the antibody titer began to drop. After removal of theclot the serum was divided into aliquots and stored at −20° C.

Upscale Culture Growth in a Bioreactor

An about 1 cm (in diameter) callus of genetically modified carrot cellscontaining the rh-GCD gene was plated onto Murashige and Skoog (MS) 9 cmdiameter agar medium plate containing 4.4 gr/l MSD medium (Duchefa), 9.9mg/l thiamin HCl (Duchefa), 0.5 mg folic acid (Sigma) 0.5 mg/l biotin(Duchefa), 0.8 g/l Casein hydrolisate (Ducifa), sugar 30 g/l andhormones 2-4 D (Sigma). The callus was grown for 14 days at 25° C.

Suspension cell culture was prepared by sub-culturing the transformedcallus in a MSD liquid medium (Murashige & Skoog (1962) containing 0.2mg/l 2,4-dichloroacetic acid), as is well known in the art. Thesuspension cells were cultivated in 250 ml Erlenmeyer flask (workingvolume starts with 25 ml and after 7 days increases to 50 ml) at 25° C.with shaking speed of 60 rpm. Subsequently, cell culture volume wasincreased to 1 L Erlenmeyer by addition of working volume up to 300 mlunder the same conditions. Inoculum of the small bio-reactor (10 L) [seeWO98/13469] containing 4 L MSD medium, was obtained by addition of 400ml suspension cells derived from two 1 L Erlenmeyer that were cultivatedfor seven days. After week of cultivation at 25° C. with 1 Lpm airflow,MDS medium was added up to 10 L and the cultivation continued under thesame conditions. After additional five days of cultivation, most of thecells were harvested and collected by passing the cell media through 80μnet. The extra medium was squeezed out and the packed cell cake wasstore at −70° C.

Further details of the bioreactor device may be found with regard toU.S. Pat. No. 6,391,638, issued on May 21, 2002 and previouslyincorporated by reference.

Protein Purification

In order to separate the medium from the insoluble GCD, frozen cell cakecontaining about 100 g wet weight cells was thawed, followed bycentrifugation of the thawed cells at 17000×g for 20 min at 4° C. Theinsoluble materials and intact cells were washed by re-suspension in 100ml washing buffer (20 mM sodium phosphate pH 7.2, 20 mM EDTA), and thenprecipitated by centrifugation at 17000 g for 20 min at 4° C. The rh-GCD(recombinant human GCD) was extracted and solubilized by homogenizationof the pellet in 200 ml extraction buffer (20 mM sodium phosphate pH7.2, 20 mM EDTA, 1 mM PMSF, 20 mM ascorbic acid, 3.8 gpolyvinylpolypyrrolidone (PVPP), 1 mM DTT and 1% Triton-x-100). Thehomogenate was then shaken for 30 min at room temperature and clarifiedby centrifugation at 17000×g for 20 min at 4° C. The pellet wasdiscarded and the pH of the supernatant was adjusted to pH 5.5 byaddition of concentrated citric acid. Turbidity generated after pHadjustment was clarified by centrifugation under the same conditionsdescribed above.

Further purification was performed by chromatography columns procedureas follows: 200 ml of clarified medium were loaded on 20 ml strongcation exchange resin (Macro-Prep high-S support, Bio-Rad) equilibratedin 25 mM sodium citrate buffer pH 5.5, packed in a XK column (2.6×20cm). The column was integrated with an AKTA (prime system (AmershamPharmacia Biotech) that allowed to monitor the conductivity, pH andabsorbency at 280 nm. The sample was loaded at 20 ml/min, afterwards thecolumn was washed with equilibration buffer (25 mM sodium citrate bufferpH 5.5) at flow rate of 12 ml/min until UV absorbency reached the baseline. Pre-elution of the rh-GCD was performed with equilibration buffercontaining 200 mM NaCl and the elution was obtained with equilibrationbuffer containing 600 mM NaCl. Fractions collected during the run weremonitored by enzyme activity assay, and tubes exhibiting enzymaticactivity (in the elution peak) were pooled. Pooled samples were diluted(1:5) in water containing 5% ethanol and pH adjusted to 6.0 with NaOH.Sample containing the rh-GCD was applied on the second XK column (1.6×20cm) packed with 10 ml of the same resin as in the previous column. Theresin in this column was equilibrate with 20 mM citrate buffer pH 6.0containing 5% ethanol. Following the sample load the column was washedwith the equilibration buffer and the GCD was eluted from the column byelution buffer (20 mM citrate buffer pH 6.0, 5% ethanol and 1M NaCl).The fractions of the absorbent peak in the elution step were pooled andapplied on a third column.

The final purification step was performed on a XK column (1.6×20 cm)packed with 8 ml hydrophobic interaction resin (TSK gel, ToyopearlPhenyl-650C, Tosoh Corp.). The resin was equilibrated in 10 mM citratebuffer pH 6.0 containing 5% ethanol. The GCD elution pool from theprevious column was loaded at 6 ml/min followed by washing withequilibration buffer until the UV absorbent reach the baseline. The pureGCD was eluted by 10 mM citric buffer containing 50% ethanol, pooled andstored at −20° C.

Determination of Protein Concentration

Protein concentrations in cell extracts and fractions were assayed bythe method of Lowry/Bradford (Bio Rad protein assay) [Bradford, M.,Anal. Biochem. (1976) 72:248] using a bovine serum albumin standard(fraction V Sigma). Alternatively, concentration of homogenous proteinsamples was determined by absorption at 280 nm, 1 mg/ml=1.4 O.D₂₈₀.Purity was determined by 280/260 nm ratio.

GCD Enzyme Activity Assay

Enzymatic activity of GCD was determined usingp-nitrophenyl-β-D-glucopyranoside (Sigma) as a substrate. Assay buffercontained 60 mM phosphate-citrate buffer pH=6, 4 mM β-mercaptoethanol,1.3 mM EDTA, 0.15% Triton X-100, 0.125% sodium taurocholate. Assay waspreformed in 96 well ELISA plates, 0-50 microliter of sample wereincubated with 250 microliter assay buffer and substrate was added tofinal concentration of 4 mM. The reaction was incubated at 37° C. for 60min. Product (p-nitrophenyl; pNP) formation was detected by absorbanceat 405 nm. Absorbance at 405 nm was monitored at t=0 and at the endpoint. After 60 min, 6 microliter of 5N NaOH were added to each well andabsorbance at 405 nm was monitored again. Reference standard curveassayed in parallel, was used to quantitate concentrations of GCD in thetested samples [Friedman et al., (1999) Blood, 93(9):2807-16].

Kinetic studies: For kinetic studies, GCD activity was assayed asdescribed by hereinabove with some modifications, using a fluorescentshort-acyl-chain analogue of glucosylceramide,N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl]-Derythro-glucosylsphingosine (C6-NBD-D-erythro-GlcCer). C6-NBD-GlcCer wassynthesized by N-acylation of glucosylsphingosine using succinimidyl6-7-nitrobenzo-2-oxa-1,3-diazol-4-yl) aminohexanoate as described bySchwarzmann and Sandhoff (1987). The assay was performed using 0.2 μg ofeither Cerezyme® or plant GCD of the invention in a final volume of 200μl MES buffer (50 mM, pH 5.5). Concentrations of C₆-NBD-GlcCer rangedfrom 0.25 to 100 μM. Reactions were allowed to proceed for 5 min at 37°C., and were stopped by addition of 1.5 ml of chloroform/methanol (1:2,v/v) prior to extraction and analysis of the fluorescent lipids.

Biochemical Analyses:

In Gel Proteolysis and Mass Spectrometry Analysis

The stained protein bands in the gel were cut with a clean razor bladeand the proteins in the gel were reduced with 10 mM DTT and modifiedwith 100 mM iodoacetamide in 10 mM ammonium bicarbonate. The gel pieceswere treated with 50% acetonitrile in 10 mM ammonium bicarbonate toremove the stain from the proteins following by drying the gel pieces.The dried gel pieces were rehydrated with 10% acetonitrile in 10 mMammonium bicarbonate containing about 0.1 μg trypsin per sample. The gelpieces were incubated overnight at 37° C. and the resulting peptideswere recovered with 60% acetonitrile with 0.1% trifluoroacetate.

The tryptic peptides were resolved by reverse-phase chromatography on0.1×300-mm fused silica capillaries (J&W, 100 micrometer ID) home-filledwith porous R2 (Persepective). The peptides were eluted using a 80-minlinear gradient of 5 to 95% acetonitrile with 0.1% acetic acid in waterat flow rate of about 1 μl/min. The liquid from the column waselectrosprayed into an ion-trap mass spectrometer (LCQ, Finnegan, SanJose, Calif.). Mass spectrometry was performed in the positive ion modeusing repetitively full MS scan followed by collision inducesdissociation (CID) of the most dominant ion selected from the first MSscan. The mass spectrometry data was compared to simulated proteolysisand CID of the proteins in the NR-NCBI database using the Sequestsoftware [J. Eng and J. Yates, University of Washington and Finnegan,San Jose].

The amino terminal of the protein was sequenced on Peptide Sequencer494A (Perkin Elmer) according to manufacture instructions.

GCD Uptake of Peritoneal Macrophages

Targeting and uptake of GCD to macrophages is known to be mediated bythe Mannose/N-acetylglucosmine receptor and can be determined usingthioglycolate-elicited peritoneal macrophages obtained from mice, asdescribed by Stahl P. and Gordon S. [J. Cell Biol. (1982) 93(1):49-56].Briefly, mice (female, strain C57-B6) were injected intraperitoneallywith 2.5 ml of 2.4% Bacto-thioglycolate medium w/o dextrose (Difco Cat.No. 0363-17-2). After 4-5 days, treated mice were sacrificed by cervicaldislocation and the peritoneal cavity rinsed with phosphate bufferedsaline. Cells were pelleted by centrifugation (1000×g 10 min) and wereresuspended in DMEM (Beit Haemek, Israel) containing 10% fetal calfserum. Cells were then plated at 1-2×10⁵ cell/well in 96-well tissueculture plates and incubated at 37° C. After 90 minutes, non-adherentcells were washed out three times using PBS, and the adherentmacrophages were incubated for 90 min at 37° C., in culture mediumcontaining specified quantities of rhGCD, ranging from 0 to 40micrograms in 200 microliter final volume, in the absence and presenceof yeast mannan (2-10, 5 mg/ml). After incubation, medium containingexcess rGCD was removed, and cells were washed three times with PBS andthen lysed with lysis buffer (10 mM Tris pH=7.3, 1 mM MgCl₂, 0.5% NP-40and protease inhibitors). The activity of rGCD taken up by the cells wasdetermined by subjecting the cell lysates to in vitro glycosidase assayas described above.

Example 1 Construction of Expression Plasmid

This Example describes the construction of an exemplary expressionplasmid, used with regard to the Examples below, in more detail.

The cDNA coding for hGCD (ATTC clone number 65696) was amplified usingthe forward: 5′ CAGAATTCGCCCGCCCCTGCA 3′ (also denoted by SEQ ID NO: 1)and the reverse: 5′ CTCAGATCTTGGCGATGCCACA 3′ (also denoted by SEQ IDNO: 2) primers.

The purified PCR DNA product was digested with endonucleases EcoRI andBglII (see recognition sequences underlined in the primers) and ligatedinto an intermediate vector having an expression cassette CE-T digestedwith the same enzymes. CE-T includes ER targeting signalMKTNLFLFLIFSLLLSLSSAEA (also denoted by SEQ ID NO: 3) from the basicendochitinase gene [Arabidopsis thaliana], and vacuolar targeting signalfrom Tobacco chitinase A: DLLVDTM* (also denoted by SEQ ID NO: 4).

The expression cassette was cut and eluted from the intermediate vectorand ligated into the binary vector pGREENII using restriction enzymesSmaI and XbaI, forming the final expression vector. Kanamycineresistance is conferred by the NPTII gene driven by the nos promotertogether with the pGREEN vector (FIG. 1B). The resulting expressioncassette is presented by FIG. 1A.

The resulting plasmid was sequenced to ensure correct in-frame fusion ofthe signals using the following sequencing primers:

Primer from the 5′ 35S promoter: 5′ CTCAGAAGACCAGAGGGC 3′ (also denotedby SEQ ID NO: 5), and the 3′ terminator: 5′ CAAAGCGGCCATCGTGC 3′ (alsodenoted by SEQ ID NO: 6). The verified cloned hGCD coding sequence isdenoted by SEQ ID NO: 7.

Example 2 Transformation of Carrot Cells and Screening for TransformedCells Expressing rhGCD

This Example describes an exemplary method for transforming carrot cellsaccording to the present invention, as used in the Examples below.

Transformation of carrot cells was performed by Agrobacteriumtransformation as described previously by [Wurtele and Bulka (1989)ibid.]. Genetically modified carrot cells were plated onto Murashige andSkoog (MS) agar medium with antibiotics for selection of transformants.As shown by FIG. 2, extracts prepared from arising calli were tested forexpression of GCD by Western blot analysis using anti hGCD antibody, andwere compared to Cerezyme standard (positive control) and extracts ofnon-transformed cells (negative control). Of the various calli tested,one callus (number 22) was selected for scale-up growth and proteinpurification.

The Western blot was performed as follows.

For this assay, proteins from the obtained sample were separated in SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose.For this purpose, SDS polyacrylamide gels were prepared as follows. TheSDS gels consist of a stacking gel and a resolving gel (in accordancewith Laemmli, UK 1970, Cleavage of structural proteins during assemblyof the head of bacteriphage T4, Nature 227, 680-685). The composition ofthe resolving gels was as follows: 12% acrylamide (Bio-Rad), 4microliters of TEMED (N,N,N′,N′-tetramethylethylenediamine; Sigmacatalog number T9281) per 10 ml of gel solution, 0.1% SDS, 375 mMTris-HCl, pH 8.8 and ammonium persulfate (APS), 0.1%. TEMED and ammoniumpersulfate were used in this context as free radical starters for thepolymerization. About 20 minutes after the initiation of polymerization,the stacking gel (3% acrylamide, 0.1% SDS, 126 mM Tris-HCl, pH 6.8, 0.1%APS and 5 microliters of TEMED per 5 ml of stacking gel solution) waspoured above the resolving gel, and a 12 or 18 space comb was insertedto create the wells for samples.

The anode and cathode chambers were filled with identical buffersolution: Tris glycine buffer containing SDS (Biorad, catalog number161-0772), pH 8.3. The antigen-containing material was treated with 0.5volume of sample loading buffer (30 ml glycerol (Sigma catalog numberG9012), 9% SDS, 15 ml mercaptoethanol (Sigma catalog number M6250),187.5 mM Tris-HCl, pH 6.8, 500 microliters bromophenol blue, all volumesper 100 ml sample buffer), and the mixture was then heated at 100° C.for 5 minutes and loaded onto the stacking gel.

The electrophoresis was performed at room temperature for a suitabletime period, for example 45-60 minutes using a constant current strengthof 50-70 volts followed by 45-60 min at 180-200 Volt for gels of 13 by 9cm in size. The antigens were then transferred to nitrocellulose(Schleicher and Schuell, Dassel).

Protein transfer was performed substantially as described herein. Thegel was located, together with the adjacent nitrocellulose, betweenWhatmann 3 MM filter paper, conductive, 0.5 cm-thick foamed material andwire electrodes which conduct the current by way of platinum electrodes.The filter paper, the foamed material and the nitrocellulose were soakedthoroughly with transfer buffer (TG buffer from Biorad, catalog number161-0771, diluted 10 times with methanol and water buffer (20%methanol)). The transfer was performed at 100 volts for 90 minutes at 4°C.

After the transfer, free binding sites on the nitrocellulose weresaturated, at 4° C. over-night with blocking buffer containing 1% drymilk (Dairy America), and 0.1% Tween 20 (Sigma Cat P1379) diluted withphosphate buffer (Riedel deHaen, catalog number 30435). The blot stripswere incubated with an antibody (dilution, 1:6500 in phosphate buffercontaining 1% dry milk and 0.1% Tween 20 as above, pH 7.5) at 37° C. for1 hour.

After incubation with the antibody, the blot was washed three times forin each case 10 minutes with PBS (phosphate buffered sodium phosphatebuffer (Riedel deHaen, catalog number 30435)). The blot strips were thenincubated, at room temperature for 1 h, with a suitable secondaryantibody (Goat anti rabbit (whole molecule) HRP (Sigma cat #A-4914)),dilution 1:3000 in buffer containing 1% dry milk (Dairy America), and0.1% Tween 20 (Sigma Cat P1379) diluted with phosphate buffer (RiedeldeHaen, catalog number 30435)). After having been washed several timeswith PBS, the blot strips were stained with ECL developer reagents(Amersham RPN 2209).

After immersing the blots in the ECL reagents the blots were exposed toX-ray film FUJI Super RX 18×24, and developed with FUJI-ANATOMIXdeveloper and fixer (FUJI-X fix cat# FIXRTU 1 out of 2). The bandsfeaturing proteins that were bound by the antibody became visible afterthis treatment.

Upscale Culture Growth in Bioreactors

Suspension cultures of callus 22 were obtained by sub-culturing oftransformed callus in a liquid medium. Cells were cultivated in shakingErlenmeyer flasks, until total volume was sufficient for inoculating thebioreactor (as described in Experimental procedures). The geneticallymodified transgenic carrot cells can be cultivated over months, and cellharvest can be obtained in cycling of 5 to 7 days (data not shown). Atthe seventh cultivation day, when the amount of rh-GCD production incarrot cell is at the peak, cells were harvested by passing of culturethrough 100 mesh nets. It should be noted that cells may be harvested bymeans known in the art such as filtration or centrifugation. The packedcell cake, which provides the material for purification of h-GCD tohomogeneity, can be stored at freezing temperature.

Example 3 Purification of recombinant active hGCD protein fromTransformed Carrot Cells

Recombinant h-GCD expressed in transformed carrot cells was found to bebound to internal membranes of the cells and not secreted to the medium.Mechanically cell disruption leaves the rGCD bound to insoluble membranedebris (data not shown). rGCD was then dissolved using mild detergents,and separated from cell debris and other insoluble components. Thesoluble enzyme was further purified using chromatography techniques,including cation exchange and hydrophobic interaction chromatographycolumns as described in Experimental procedures.

In order to separate the medium from the insoluble GCD, frozen cell cakecontaining about 100 g wet weight cells was thawed, followed bycentrifugation at 17000×g for 20 min at 4° C. The insoluble materialsand intact cells were washed by re-suspension in 100 ml washing buffer(20 mM sodium phosphate pH 7.2, 20 mM EDTA), and precipitated bycentrifugation at 17000 g for 20 min at 4° C. The rGCD was extracted andsolubilized by homogenization of the pellet in 200 ml extraction buffer(20 mM sodium phosphate pH 7.2, 20 mM EDTA, 1 mM PMSF, 20 mM ascorbicacid, 3.8 g polyvinylpolypyrrolidone (PVPP), 1 mM DTT, 1% Triton-x-100(Sigma)). The homogenate was shaken for 30 min at room temperature andclarified by centrifugation at 17000 g for 20 min at 4° C. The pelletwas discarded and the pH of the supernatant was adjusted to pH 5.5 byaddition of concentrated citric acid. Turbidity generated after pHadjustment was clarified by centrifugation under the same conditionsdescribed above.

Further purification was performed by chromatography columns as follows:in a first stage, 200 ml of clarified extract were loaded on 20 mlstrong cation exchange resin (Macro-Prep high-S support, Bio-Rad)equilibrated in 25 mM sodium citrate buffer pH 5.5, packed in a XKcolumn (2.6×20 cm). The column was integrated with an AKTA prime system(Amersham Pharmacia Biotech) that allowed to monitor the conductivity,pH and absorbency at 280 nm. The sample was loaded at 20 ml/min,afterwards the column was washed with equilibration buffer (25 mM sodiumcitrate buffer pH 5.5) at flow rate of 12 ml/min until UV absorbencyreached the base line. Pre-elution of the rh-GCD was performed withequilibration buffer containing 200 mM NaCl and the elution was obtainedwith equilibration buffer containing 600 mM NaCl. Fractions collectedduring the run were monitored by enzyme activity assay, and tubesexhibiting enzymatic activity (in the elution peak) were pooled. Pooledsamples were diluted (1:5) in water containing 5% ethanol and pHadjusted to 6.0 with NaOH.

FIG. 3A represents a standard run of this purification stage. Thefractions collected during the run were monitored by enzyme activityassay, as shown by FIG. 3B, and FIG. 3C shows coomassie-blue stain ofelution fractions assayed for activity.

Elution fractions containing the rGCD was applied on a second XK column(1.6×20 cm) packed with 10 ml of the same resin as in the previouscolumn, for a second purification stage. The resin in this column wasequilibrated with 20 mM citrate buffer pH 6.0 containing 5% ethanol.Following the sample load the column was washed with the equilibrationbuffer and the rGCD was eluted from the column by elution buffer (20 mMcitrate buffer pH 6.0, 5% ethanol and 1M NaCl). FIG. 3D represents astandard run of this purification stage. The fractions collected duringthe run were monitored by enzyme activity assay, as shown by FIG. 3E,and FIG. 3F shows a coomassie-blue stain of elution fractions assayedfor activity.

The fractions of the absorbent peak in the elution step were pooled andapplied on a third column, for a third purification stage. The thirdpurification stage was performed on a XK column (1.6×20 cm) packed with8 ml hydrophobic interaction resin (TSK gel, Toyopearl Phenyl-650C,Tosoh Corp.). The resin was equilibrated in 10 mM citrate buffer pH 6.0containing 5% ethanol. The GCD elution pool from the previous column wasloaded at 6 ml/min followed by washing with equilibration buffer untilthe UV absorbance reached the baseline. The pure GCD was eluted by 10 mMcitric buffer containing 50% ethanol, pooled and stored at −20° C.

FIG. 4A represents a standard run of this purification stage. Thefractions collected during the run were monitored by enzyme activityassay (FIG. 4B), and FIG. 4C shows coomassie-blue stain of elutionfractions assayed for activity.

In a batch purification of cells that were processed, rGCD protein waspurified to a level greater than 95%; if only the first and third stagesare performed, purity is achieved at a level of about 80% (results notshown).

Biochemical Analysis

To validate the identity of purified rhGCD, Mass-Spec Mass-Spec (MSMS)analysis was preformed. Results obtained showed 49% coverage of proteinsequence that matched the predicted amino acid sequence, based on theDNA of the expression cassette, including the leader peptide andtargeting sequences.

Characterization and Sequencing of prGCD: To further characterize theplant produced human recombinant GCD of the invention, the rhGCD wassolubilized using Triton X-100, in the presence of an antioxidant, andpurified to homogeneity by cation exchange and hydrophobicchromatography (FIG. 9 a). Amino-acid sequencing of the plant producedhuman recombinant GCD of the invention demonstrated that the rhGCDsequence (SEQ ID NO: 15) corresponds to that of the human GCD (SwissProt P04062, protein ID AAA35873), and includes two additional aminoacids (EF) at the N-terminus (designated −2 and −1 accordingly), derivedfrom the linker used for fusion of the signal peptide, and an additional7 amino acids at the C-terminus (designated 497-503) derived from thevacuolar targeting signal.

Immunodetection of the purified plant produced human recombinant GCD ofthe invention with anti-GCD polyclonal antibody was performed by Westernblotting of the SDS-PAGE separated protein, along with Cerezyme® protein(FIG. 9 b), confirming antigenic identity of the plant produced andCHO-produced proteins.

Enzymatic Activity of Recombinant hGCD:

The activity of plant produced human recombinant GCD of the presentinvention was compared to that of Cerezyme®, using a fluorescent GlcCeranalogue. FIG. 12 shows that similar specific activities were obtained,with V_(max) values of 0.47±0.08 Kmol C6-NBD-ceramide formed/min/mgprotein for prGCD and 0.43±0.06 for Cerezyme®, and similar K_(m) values(20.7±0.7 KM for the GCD of the invention and 15.2±4.8 KM forCerezyme®). Thus, these kinetic studies show that the activity of theplant produced human recombinant GCD of the present invention is similarto that of the CHO expressed enzyme.

Uptake and Activity of Recombinant hGCD in Peritoneal Macrophages

To determine whether the rhGCD produced in carrot has been correctlyglycosylated and can undergo uptake by target cells, and thus be usefulfor treatment of Gaucher's disease, the ability of the rhGCD to bind toand be taken up by macrophages was next assayed. Targeting of rhGCD tomacrophages is mediated by the Mannose/N-acetylglucosamine (Man/GlcNAc)receptor and can be determined using thioglycolate-elicited peritonealmacrophages. As shown by FIG. 5, rGCD undergoes uptake by cells at ahigh level. FIG. 5A shows uptake by cells of rGCD according to thepresent invention with regard to mannan concentration.

FIG. 5A shows uptake at comparable levels with Cerezyme™ (thispreparation was prepared to 80% purity with only the first and thirdstages of the purification process described above).

FIGS. 5B and 5C show that rGCD uptake is at a higher level thanCerezyme™, as this preparation was prepared to greater than 95% puritywith all three stages of the purification process described above.

With regard to FIG. 5C, clearly the percent of specific activity fromtotal activity, inhibited by 4 mg/ml mannan, is higher for the GCD ofthe present invention (rGCD or recombinant human GCD) than for thecurrently available product in the market as follows: GCD (CB-mix1,which is the rGCD of the present invention)—75% Cerezyme -65%.Furthermore, as shown by the figures, addition of mannan clearlyinhibited binding of rGCD by the cells. At concentration of 2 mg/ml ofmannan, the binding of rGCD was inhibited by 50%.

These results show that even without remodeling of glycan structures,rhGCD expressed and purified from transformed carrot cells can undergouptake to target macrophage cells specifically through Man/GlcNAcreceptors. Moreover, this recombinant rhGCD is enzymatically active.

FIG. 5D shows that the rhGCD is also recognized by an anti-GCD antibodyin a Western blot; rGCD refers to the protein according to the presentinvention, while GCD standard (shown at 5, 10 and 25 ng per lane) iscommercially purchased GCD (Cerezyme®).

Example 4 Toxicology Testing

The material obtained according to the above purification procedure wastested according to standard toxicology testing protocols (Guidance forIndustry on Single Dose Acute Toxicity Testing for Pharmaceuticals,Center for Drug Evaluation and Research (CDER) PT 1 (61 FR 43934, Aug.26, 1996) and by ICH M3(M) Non-clinical Safety Studies for the Conductof Human Clinical Trials for Pharmaceuticals CPMP/ICH/286/95modification, Nov. 16, 2000).

Mice were injected as follows: An initial dose of 1.8 mg/kg (clinicaldose) was followed by doses of 9 and 18 mg/kg. Testing groups includedsix mice (ICR CD-1; 3 males and 3 females) for receiving rGCD (in aliquid carrier featuring 25 mM citrate buffer, 150 mM NaCl, 0.01% Tween80, 5% ethanol) according to the present invention, and another six micefor being treated with the carrier alone as a control group. The micewere then observed for 14 days and were euthanized.

In another study, vehicle solution alone, or doses of prGCD in multiplesof 1, 5, or 10 times the standard clinical dose (60 units/kg) were givento ICR (CD-1®) mice. The animals (6 per group, 3 males and 3 females),received the drug intravenously in a 10 ml/kg volume.

Both toxicity studies revealed no obvious treatment-related adversereactions, no gross pathological findings, no changes in body weight andno mortality incidences observed even at the highest dose administered.Furthermore, blood samples taken from animals in the high-dose group,which had been administered with 10-fold the clinical dose, were testedfor hematology and clinical chemistry. All hematology and clinicalchemistry values were in normal ranges. In addition, the animals treatedwith the high dose were subjected to histopathological examination ofthe liver, spleen and kidney, and there were no macro or microhistopathological findings.

Example 5 Glycosylation Analysis

Analysis of glycan structures present on rGCD produced as described withregard to the previous Examples was performed. As described in greaterdetail below, results indicate that the majority of glycans containterminal mannose residues as well as high mannose structures.Advantageously, this high mannose product was found to be biologicallyactive, and therefore no further steps were needed for its activation.

The following methods were used to determine the glycosylation structureof the recombinant hGCD produced according to the Examples given above.Briefly, the monosaccharide linkages for both N- and O-glycans weredetermined by using a hydrolysis and GC-MS strategy. This methodestimates the linkage type of the carbohydrates to the peptide and thegeneral monosaccharide composition of a glycoprotein. Based on priorknowledge and also the ratios between various monosaccharides, thismethod may suggest the types of glycans on the glycoprotein. Thisinformation is important to estimate the possible glycan structurespresent on the protein.

Another method featured oligosaccharide analysis of the N-glycanpopulation. FAB-MS and MALDI-TOF MS were performed, following digestionof aliquots of the samples with trypsin and peptide N-glycosidase F(PNGaseF) and permethylation of the glycans. This method is used todetach and isolate N-linked carbohydrates from the enzymaticallydigested glycoprotein. The masses of the glycan populations in theisolated glycan mix are determined and their masses are compared withthose of known structures from databases and in light of themonosaccharide composition analysis. The proposed structures are basedalso on the glycosylation patterns of the source organism.

Another method included analyzing the O-glycan population followingreductive elimination of the tryptic and PNGase F treated glycopeptides,desalting and permethylation. O-glycans are not released by PNGase F,therefore, glycans remaining linked to peptides are most likely O-linkedglycans. These glycans are then released by reductive elimination andtheir mass analyzed.

Monosaccharide composition analysis (summarized below) revealed acharacteristic distribution of hexoses, hexosamines and pentosescharacteristic of plant glycosylation. The ratios between GlcNac andMannose, suggest that characteristic N-linked structures are thepredominant glycan population.

Mass Spectrometric analysis of the N-glycans from hGCD produced asdescribed above indicates that the predominant N-glycan population hasthe monosaccharide composition Pent.deoxyHex.Hex3.HexNAc2.

Materials and Methods

Analysis was performed using a combination of Gas Chromatography-MassSpectrometry (GC-MS), Fast Atom Bombardment-Mass Spectrometry (FAB-MS)and Delayed Extraction-Matrix Assisted Laser Desorption Ionisation-Timeof Flight Mass-Spectrometry (DE-MALDI-TOF MS).

For oligosaccharide analysis, the N-glycan population was analysed byFAB-MS and MALDI-TOF MS following digestion of aliquots of the sampleswith trypsin and peptide N-glycosidase F (PNGaseF) and permethylation ofthe glycans. The O-glycan population was analysed following reductiveelimination of the tryptic and PNGase F treated glycopeptides, desaltingand permethylation.

The monosaccharide linkages for both N- and O-glycans were determinedusing a hydrolysis, derivatisation GC-MS strategy.

Experimental Description

Sample

The sample vials were received were given the unique sample numbers asfollows (Table 1):

TABLE 1 reference Product number Glucocerebrosidase. Four tubescontaining 62995 1 ml of sample each at a stated 62996 concentration of0.8 mg/ml in 25 mM 62997 Citrate Buffer pH 6.0, 0.01% Tween 80 62998

The samples were stored between −10 and −30° C. until required.

Protein Chemistry

Dialysis of Intact Samples

One vial (containing 1 ml of protein at a stated concentration of 0.8mg/ml) was injected into a Slide-A-Lyzer dialysis cassette (10 kDamolecular weight cutoff) and dialysed at 4° C. over a period of 24 hoursagainst water, the water being changed 3 times. Following dialysis thesample was removed form the cassette and lyophilised.

Trypsin Digestion of the Intact Samples for Oligosaccharide Screening

The dialysed, lyophilised sample was resuspended in 50 mM ammoniumbicarbonate buffer adjusted to pH 8.4 with 10% aq. ammonia and digestedwith TPCK treated trypsin for 4 hours at 37° C. according to SOPs B001and B003. The reaction was terminated by placing in a heating block at95° C. for 2 minutes followed by lyophilisation.

Carbohydrate Chemistry Peptide N-Glycosidase A Digestion

The tryptically cleaved peptide/glycopeptide mixtures from theglycoprotein sample was treated with the enzyme peptide N-glycosidase A(PNGaseA) in ammonium acetate buffer, pH 5.5 at 37° C. for 15 hours. Thereaction was stopped by freeze-drying. The resulting products werepurified using a C₁₈ Sep-Pak cartridge.

Reductive Elimination

The Sep-Pak fraction containing potential O-linked glycopeptides wasdissolved in a solution of 10 mg/ml sodium borohydride in 0.05M sodiumhydroxide and incubated at 45° C. for 16 hours. The reaction wasterminated by the addition of glacial acetic acid.

Desalting of Reductively Eliminated Material

Desalting using Dowex beads was performed according to SOP B022. Thesample was loaded onto the column and eluted using 4 ml of 5% aq. aceticacid. The collected fraction was lyophilised.

Permethylation of Released Carbohydrates

N-linked carbohydrates eluting in the 5% aq. acetic acid Sep-Pakfraction and potential O-linked glycans released by reductiveelimination, were permethylated using the sodium hydroxide (NaOH)/methyliodide (MeI) procedure (SOP B018). A portion of the permethylatedN-linked glycan mixture was analysed by FAB-MS and MALDI-TOF MS and theremainder was subjected to linkage analysis.

Linkage Analysis of the N-linked Carbohydrate

Derivatisation

The permethylated glycan sample mixtures obtained following tryptic andPNGase A digestion or reductive elimination were hydrolysed (2M TFA, 2hours at 120° C.) and reduced (sodium borodeuteride (NaBD₄) in 2M NH₄OH,2 hours at room temperature, SOP B025). The borate produced on thedecomposition of the borodeuteride was removed by 3 additions of amixture of methanol in glacial acetic acid (90:10) followed bylyophilisation. The samples were then acetylated using acetic anhydride(1 hour at 100° C.). The acetylated samples were purified by extractioninto chloroform. The partially methylated alditol acetates were thenexamined by gas chromatography/mass spectrometry (GC/MS). Standardmixtures of partially methylated alditol acetates and a blank were alsorun under the same conditions.

Gas Liquid Chromatography/Mass Spectrometry (GC/MS)

An aliquot (1 μl) of the derivatised carbohydrate samples dissolved inhexane, were analysed by GC/MS using a Perkin Elmer Turbomass Gold massspectrometer with an Autosystem XL gas chromatograph and a Dell datasystem under the following conditions:

Gas Chromatography

Column: DB5

Injection: On-column

Injector Temperature: 40° C.

Programme: 1 minute at 40° C. then 70° C./minute to 100° C., held at100° C. for 1 minute, then 8° C./minute to 290° C., finally held at 290°C. for 5 minutes.

Carrier Gas: Helium

Mass Spectrometry

Ionisation Voltage: 70 eV

Acquisition Mode Scanning

Mass Range: 35-450 Daltons

MS Resolution: Unit

Sugar Analysis of Intact Glucocerebrosidase

Derivatisation

An aliquot equivalent to 500 μg of glucocerebrosidase was lyophilisedwith 10 μg of Arabitol as internal standard. This was then methanolysedovernight at 80° C. and dried under nitrogen. Released monosaccharideswere re-N-acetylated using a solution of methanol, pyridine and aceticanhydride, dried under nitrogen again and converted to theirtrimethylsilyl (TMS) derivatives according to SOP B023. The TMSderivatives were reduced in volume under nitrogen, dissolved in 2 ml ofhexane and sonicated for 3 minutes. The samples were then allowed toequilibrate at 4° C. overnight. A blank containing 10 μg of Arabitol anda standard monosaccharide mixture containing 1 μg each of Fucose,Xylose, Mannose, Galactose, Glucose, N-acetylgalactosamine,N-acetylglucosamine, N-acetylneuraminic acid and Arabitol were preparedin parallel. The TMS derivatives were then examined by gaschromatography/mass spectrometry (GC/MS).

Gas Liquid Chromatography/Mass Spectrometry

(GC/MS)

An aliquot (1 μl) of the derivatised carbohydrate sample dissolved inhexane, was analysed by GC/MS using a Perkin Elmer Turbomass Gold massspectrometer with an Autosystem XL gas chromatograph and a Dell datasystem under the following conditions:

Gas Chromatography

Column: DB5

Injection: On-column

Injector Temperature: 40° C.

Programme: 1 minute at 90° C. then 25° C./minute to 140° C., 5°C./minute to 220° C., finally 10° C./minute to 300° C. and held at 300°C. for 5 minutes.

Carrier Gas: Helium

Mass Spectrometry

Ionisation Voltage: 70 eV

Acquisition Mode: Scanning

Mass Range: 50-620 Daltons

MS Resolution: Unit

Delayed Extraction Matrix Assisted Laser Desorption Ionisation MassSpectrometry (DE-MALDI-MS) and Fast Atom Bombardment-Mass Spectrometry(FAB-MS)

MALDI-TOF mass spectrometry was performed using a Voyager STRBiospectrometry Research Station Laser-Desorption Mass Spectrometercoupled with Delayed Extraction (DE).

Dried permethylated glycans were redissolved in methanol:water (80:20)and analysed using a matrix of 2,5-dihydroxybenzoic acid. Bradykinin,Angiotensin and ACTH were used as external calibrants.

Positive Ion Fast Atom Bombardment mass spectrometric analyses werecarried out on M-Scan's VG AutoSpecE mass spectrometer operating atVacc=8 kV for 4500 mass range at full sensitivity with a resolution ofapproximately 2500. A Caesium Ion Gun was used to generate spectraoperating at 30 kV. Spectra were recorded on a VAX data system 3100 M76using Opus software.

Dried permethylated glycans were dissolved in methanol and loaded onto atarget previously smeared with 2-4 μl of thioglycerol as matrix prior toinsertion into the source.

In a second set of glycosylation analysis, similar methods were used todetermine the glycosylation patterns, and to identify the majorglycosylated products produced by the carrot cell suspension culture ofthe present invention:

Glycosylation patterns were analyzed by the Glycobiology Center of theNational Institute for Biotechnology (Ben Gurion University, Beer Sheba,Israel) to determine glycan structure and relative amounts usingsequential digestion with various exoglycosidases. The plant GCD samplesof the invention were run on SDS-PAGE and a 61 KDa band was cut out andincubated with either PNGase A, or with trypsin followed by PNGase A torelease the N-linked glycans. The glycans were fluorescently labeledwith anthranilamide (2AB) and run on normal phase HPLC.

Sequencing of the labeled glycan pool was achieved by sequentialdigestion with various exoglycosidases followed by HPLC analysis.Retention times of individual glycans were compared to those of astandard partial hydrolysate of dextran giving a ladder of glucose units(GU). Unlabeled glycans were further purified and analyzed by MALDI massspectrometry. Exoglycosidases used: Bovine kidney-fucosidase (digests1-6 and 1-3 core fucose, Prozyme), Jack bean mannosidase (removes 1-2,6>3 mannose, Prozyme), Xanthomonas beta-1,2-xylosidase (removes 1-2xylose only after removal of -linked mannose, Calbiochem).

Bovine testes-galactosidase (hydrolyses non-reducing terminal galactose1-3 and 1-4 linkages, Prozyme), Streptococcus pneumoniae hexosaminidase(digest 1-2, 3, 4, 6 GalNAc and GlcNAc, Prozyme). Glycosylation wasfurther analyzed by M-Scan (Berkshire, England) using gas chromatographymass spectrometry (GC-MS), fast atom bombardment-mass spectrometry(FAB-MS), and delayed extraction-matrix assisted laser desorptionionization—time of flight mass-spectrometry (DE-MALDI-TOF MS). Foroligosaccharide determination, the N-glycan population was analyzed byFAB-MS and MALDI-TOF MS, following digestion of samples with trypsin andPNGase A, and permethylation of the glycans. O-glycans were analyzedfollowing reductive elimination of the tryptic and PNGase A-treatedglycopeptides, desalting and permethylation.

The similarity of the N-glycans in different batches of prGCD wasanalyzed by high performance anion exchange chromatography with pulsedamperometric detection (HPAEC-PAD, a Dionex method) following digestionwith trypsin and PNGase A, to obtain chromatographic profiles foroligosaccharides released from glycoproteins for the purpose ofdemonstrating consistency from batch to batch of prGCD. This procedurepermits chromatographic comparison of oligosaccharide patterns in aqualitative and quantitative manner.

Results and Discussion

TMS Sugar Analysis of Glucocerebrosidase N-Linked OligosaccharideScreening

The intact glycoprotein was subjected to dialysis followed by trypsindigestion and the lyophilised products were digested using PNGase A andthen purified using a C₁₈ Sep-Pak. The 5% aq. acetic acid (N-linkedoligosaccharide containing) fraction was permethylated and FAB massspectra were obtained using a portion of the derivatised oligosaccharidein a low mass range for fragment ions and DE-MALDI-TOF mass spectra wereobtained using a portion of the derivatised oligosaccharides in a highmass range for molecular ions.

Analysis of N-Glycans from Glucocerebrosidase

Table 1 lists the predominant fragment ions present in the FAB spectraand molecular ions present in the MALDI spectra. The molecular ionregion (shown in Appendix III) contains a predominant signal at m/z1505.8 (consistent with an [M+Na]⁺ quasimolecular ion for a structurehaving the composition Pent.deoxyHex.Hex₃.HexNAc₂). A range of lessintense quasimolecular ions were also detected consistent with complexand high mannose structures. The high mannose structures detected rangein size from Hex₅.HexNAc₂ at m/z 1579.8 to Hex₈.HexNAc₂ at m/z 2193.0.The complex signals are produced from less extensively processedN-glycans such as m/z 1331.7 (consistent with an [M+Na]⁺ quasimolecularion for a structure having the composition Pent.Hex₃.HexNAc₂) or fromlarger N-glycans for example m/z 1751.0 (consistent with an [M+Na]⁺quasimolecular ion for a structure having the compositionPent.deoxyHex.Hex₃.HexNAc₃), m/z 2375.4 (consistent with an [M+Na]⁺quasimolecular ion for a structure having the compositionPent.deoxyHex₂.Hex4.HexNAc₄) and m/z 2753.6 (consistent with an [M+Na]⁺quasimolecular ion for a structure having the compositionPent.deoxyHex₃.Hex₅.HexNAc₄).

The FAB mass spectrum provides information regarding antennae structuresby virtual of fragment ions in the low mass region of the spectrum (datanot shown). Signals were detected identifying hexose (at m/z 219) andHexNAc (at m/z 260) as non-reducing terminal monosaccharides in theN-glycans.

TABLE 2 Masses observed in the permethylated spectra ofGlucocerebrosidase (reference number 62996) following Tryptic andPeptide N-glycosidase A digestion Signals observed (m/z) PossibleAssignment Low Mass 219 Hex⁺ 228 HexNAc⁺ (−methanol) 260 HexNAc⁺ HighMass 1032.4 Pent•Hex₃•HexNAc⁺ 1171.5 Hex₃•HexNAc₂OMe + Na⁺ 1299.6Elimination of fucose from m/z 1505.8 1331.6 Pent•Hex₃•HexNAc₂OMe + Na⁺1345.6 deoxyHex•Hex₃•HexNAc₂OMe + Na⁺ 1505.7Pent•deoxyHex•Hex₃•HexNAc₂OMe + Na⁺ 1579.8 Hex₅•HexNAc₂OMe + Na⁺ 1709.9Pent•deoxyHex•Hex₄•HexNAc₂OMe + Na⁺ 1750.9Pent•deoxyHex•Hex₃•HexNAc₃OMe + Na⁺ 1783.9 Hex₆•HexNAc₂OMe + Na⁺ 1989.0Hex₇•HexNAc₂OMe + Na⁺ 1997.0 Pent•deoxyHex•Hex₃•HexNAc₄OMe + Na⁺ 2027.0Not assigned 2099.0 Not assigned 2130.0 Pent•deoxyHex₂•Hex₄•HexNAc₃OMe +Na⁺ 2193.1 Hex₈•HexNAc₂OMe + Na⁺ 2375.2 Pent•deoxyHex₂•Hex₄•HexNAc₄OMe +Na⁺ 2753.4 Pent•deoxyHex₃•Hex₅•HexNAc₄OMe + Na⁺

All masses in column one are monoisotopic unless otherwise stated. Themass numbers may not relate directly to the raw data as the softwareoften assigns mass numbers to ¹³C isotope peaks particularly for massesabove 1700Da.

Linkage Analysis of N-Glycans from Glucocerebrosidase

Linkage analysis was performed on the N-linked carbohydrates releasedfollowing PNGase A digestion, Sep-Pak purification and permethylation.

A complex chromatogram was obtained with some impurity peaks originatingfrom the derivatising reagents. Comparison of the retention time and thespectra with standard mixtures allowed provisional assignments of thesugar containing peaks listed in Table 3.

TABLE 3 Retention times of the variously linked monosaccharides detectedas their partially methylated alditol acetates in the GC-MS analysis ofGlucocerebrosidase (reference number 62996) following Tryptic andPeptide N-glycosidase A digestion Retention time Compounds (mins)Glucocerebrosidase Observed (62996) Terminal 10.41 Xylose Terminal 10.84Fucose Terminal 12.29 (major) Mannose Terminal 12.55 Galactose 2-linked13.40 Mannose 4-linked 13.58 Glucose 2,6-linked 14.91 Mannose 3,6-linked15.08 Mannose 2,3,6-linked 15.87 Mannose 4-linked 16.73 GlcNAc3,4-linked 17.59 GlcNAc

4.3 O-Linked Oligosaccharide Screening

Reductive elimination was carried out on the 60% 2-propanol fraction(potential O-linked glycopeptide fraction) from the Sep-Pak purificationof Glucocerebrosidase following trypsin and PNGase A digestions. Thesample was desalted following termination of the reaction and, afterborate removal, was permethylated. FAB mass spectra were obtained usinga portion of the derivatised oligosaccharide in a low mass range forfragment ions and DE-MALDI-TOF mass spectra were obtained using aportion of the derivatised oligosaccharides in a high mass range formolecular ions. No signals consistent with the presence of O-linkedglycans were observed (data not shown).

Linkage Analysis of O-Glycans from Glucocerebrosidase

Linkage analysis was carried out on the products of reductiveelimination after permethylation. No signals consistent with thepresence of typical O-linked glycans were observed (data not shown).

FIG. 6 shows some exemplary glycan structures as a comparison betweenGCD obtained from CHO (Chinese hamster ovary) cells, which are mammaliancells (Cerezyme™) and the GCD of the present invention, from carrotcells. As shown, remodeling of these structures is required to obtainexposed mannose residues for Cerezyme™. By contrast, such exposedmannose residues are directly obtained for the GCD obtained from plantcells according to the present invention, without requiring furthermanipulation, for example with glycosylases.

FIG. 7 represents the main glycan structure found in rGCD. FIG. 7 showsproposed structures of: a) the predominant oligosaccharide populationfound on hGC expressed in carrot cell suspension (1505.7 m/z); b)typical N-linked core; c) Fucosylated plant N-linked core. N-linkedglycans are coupled to the protein via-Aspargine and through thereducing end of the GlcNac (GN) residue on the right hand of thediagrams. N plant glycosylation patterns, Fucose residues may be part ofthe core structure, bound to the first GlcNac using an alpha(1-3)glycosidic bond, while mammalian structures typically use the alpha(1-6)glycosidic bond.

FIGS. 8A-8D show all possible structures for the N-glycans detected onthe rGCD protein according to the present invention.

The dominant glycan structure that was identified is the core glycanstructure found in most plant glycoproteins from pea, rice, maize andother edible plants. This structure contains a core xylose residue aswell as a core alpha-(1,3)-fucose. Work done by Bardor et al (33) showsthat 50% of nonallergic blood donors have specific antibodies for corexylose in their sera, and 25% have specific antibodies to corealpha-(1,3)-fucose. However it is still to be studied whether suchantibodies might introduce limitations to the use of plant-derivedbiopharmaceutical glycoproteins.

The minor glycan populations of the hGCD produced as described abovewere mainly high mannose structures Hex4HexNAc2 to Hex8HexNAc2. Amongthe complex structures exhibited structures such asPent.deoxyHex2.Hex4.HexNAc3 and Pent.deoxyHex3.Hex5.HexNAc3.Pent.Hex3.HexNAc2 was detected in smaller proportions.

The major terminal monosaccharides are hexose (Mannose or Galactose) andN-acetylhexosamine, which is consistent with the presence of highmannose structures and partially processed complex structures.

With regard to O-linked oligosaccharide screening, no signals that areconsistent with typical O-linked glycans were observed. GCD is known inthe art to not have O-linked oligosaccharides, such that these resultsare consistent with the known glycosylation of GCD from other cellsystems, including native GCD and recombinant GCD produced in mammalianculture systems. However, in the monosaccharide composition, signalsconsistent with Arabinose were detected.

An important point with regard to the present invention is that the hGCDprotein N-glycan composition analysis showed that the majority of theN-glycans terminate with mannose residues. This agrees with therequirement for mannose terminating N-glycans assisting the uptake oftherapeutic hGCD by the macrophage mannose receptor. However, neithernative GCD nor recombinant GCD produced in mammalian cells is highmannose. Therefore, the present invention overcomes a significantdrawback of commercially produced hGCD proteins, which is that theseproteins are modified to terminate with mannose sugars, unlike theprotein produced as described above.

Further glycosylation analysis was performed on a purified humanrecombinant glucocerebrosidase prepared in plant cells. Glycosylationwas analyzed (Glycobiology Center of the National Institute forBiotechnology (Ben Gurion University, Beer Sheba, Israel) to determineglycan structure and the glycan quantitative ratio using sequentialdigestion with various exoglycosidases (see Methods, above). In thisanalysis, it was found that the N-linked glycans have a main core of twoGlcNAc residues and a 1-4 linked mannose, attached to two additionalmannose residues in 1-3 and 1-6 linkages. The additional residues foundare shown in FIG. 10 a, which presents all structures and their relativeamounts based upon HPLC, enzyme array digests and MALDI. FIG. 10 b showsthe glycan structure of Cerezyme® before and after in vitro enzymaticprocessing. Notably, analysis of the glycan structures of the GCD of theinvention revealed that >90% of the glycans were mannose-rich, bearingexposed, terminal mannose residues (FIG. 10 a), whereas in the case ofCerezyme®, mannose residues are exposed only after a complex in-vitroprocedure (FIG. 10 b). The dominant glycan in the GCD of the inventionis the core structure found in most glycoproteins purified from pea,rice, maize and other edible plants. This structure contains acore-(1,2)-xylose residue as well as a core-(1,3)-fucose (FIG. 10 a).The DE-MALDI-MS data contained no signals consistent with typicalO-linked glycans. Further analysis of the glycan profiles for the GCD ofthe invention obtained from different production batches was performedin order to assess the batch-to-batch reproducibility of the GCDproduced in the carrot cell system. As presented in FIG. 11, thepopulation of glycans on plant GCD of the invention is highlyreproducible between batches.

Example 6 Treatment with the Present Invention

The recombinant protein produced according to the present inventionpreferably comprises a suitably glycosylated protein produced by a plantcell culture, which is preferably a lysosomal enzyme for example, and/ora high mannose glycosylated protein.

According to preferred embodiments herein, the protein producedaccording to the present invention is suitable for treatment of alysosomal-associated disease, such as a lysosomal storage disease forexample.

The method of treatment optionally and preferably comprises: (a)providing a recombinant biologically active form of lysosomal enzymepurified from transformed plant root cells, and capable of efficientlytargeting cells abnormally deficient in the lysosomal enzyme. Thisrecombinant biologically active enzyme has exposed terminal mannoseresidues on appended oligosaccharides; and (b) administering atherapeutically effective amount of the recombinant biologically activelysosomal enzyme, or of composition comprising the same to the subject.In a preferred embodiment, the recombinant high mannose lysosomal enzymeused by the method of the invention may be produced by the host cell ofthe invention. Preferably, this host cell is a carrot cell.

By “mammalian subject” or “mammalian patient” is meant any mammal forwhich gene therapy is desired, including human, bovine, equine, canine,and feline subjects, most preferably, a human subject.

It should be noted that the term “treatment” also includes ameliorationor alleviation of a pathological condition and/or one or more symptomsthereof, curing such a condition, or preventing the genesis of such acondition.

In another preferred embodiment, the lysosomal enzyme used by the methodof the invention may be a high mannose enzyme comprising at least oneoligosaccharide chain having an exposed mannose residue. Thisrecombinant enzyme can bind to a mannose receptor on a target cell in atarget site within a subject. More preferably, this recombinantlysosomal enzyme has increased affinity for these target cell, incomparison with the corresponding affinity of a naturally occurringlysosomal enzyme to the target cell. Therefore, each dose is dependenton the effective targeting of cells abnormally deficient in GCD and eachdose of such form of GCD is substantially less than the dose ofnaturally occurring GCD that would otherwise be administered in asimilar manner to achieve the therapeutic effect.

According to preferred embodiments of the present invention, the proteinis suitable for the treatment of lysosomal storage diseases, such thatthe present invention also comprises a method for treating suchdiseases. Lysosomal storage diseases are a group of over 40 disorderswhich are the result of defects in genes encoding enzymes that breakdown glycolipid or polysaccharide waste products within the lysosomes ofcells. The enzymatic products, e.g., sugars and lipids, are thenrecycled into new products. Each of these disorders results from aninherited autosomal or X-linked recessive trait which affects the levelsof enzymes in the lysosome. Generally, there is no biological orfunctional activity of the affected enzymes in the cells and tissues ofaffected individuals. In such diseases the deficiency in enzyme functioncreates a progressive systemic deposition of lipid or carbohydratesubstrate in lysosomes in cells in the body, eventually causing loss oforgan function and death. The genetic etiology, clinical manifestations,molecular biology and possibility of the lysosomal storage diseases aredetailed in Scriver et al. [Scriver et al. eds., The Metabolic andMolecular Basis of Inherited Disease, 7^(th) Ed., Vol. II, McGraw Hill,(1995)].

Examples of lysosomal storage diseases (and their associated deficientenzymes) include but are not limited to Fabry disease (α-galactosidase),Farber disease (ceramidase), Gaucher disease (glucocerebrosidase),G_(ml) gangliosidosis (β-galactosidase), Tay-Sachs disease(β-hexosaminidase), Niemann-Pick disease (sphingomyelinase), Schindlerdisease (α.-N-acetylgalactosaminidase), Hunter syndrome(iduronate-2-sulfatase), Sly syndrome (β-glucuronidase), Hurler andHurler/Scheie syndromes (iduronidase), and I-Cell/San Filipo syndrome(mannose 6-phosphate transporter).

Gaucher disease is the most common lysosomal storage disease in humans,with the highest frequency encountered in the Ashkenazi Jewishpopulation. About 5,000 to 10,000 people in the United States areafflicted with this disease [Grabowski, Adv. Hum. Genet. 21:377-441(1993)]. Gaucher disease results from a deficiency in glucocerebrosidase(hGCD; glucosylceramidase). This deficiency leads to an accumulation ofthe enzyme's substrate, glucocerebroside, in reticuloendothelial cellsof the bone marrow, spleen and liver, resulting in significant skeletalcomplications such as bone marrow expansion and bone deterioration, andalso hypersplenism, hepatomegaly, thrombocytopenia, anemia and lungcomplications [Grabowski, (1993) ibid.; Lee, Prog. Clin. Biol. Res.95:177-217 (1982)].

More specifically, the lysosomal enzyme used by the method of theinvention may be selected from the group consisting ofglucocerebrosidase (GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. Preferably, where the treated disease is Gaucher'sdisease, the lysosomal enzyme used by the method of the invention isglucocerebrosidase (GCD).

The protein of the present invention can be used to produce apharmaceutical composition. Thus, according to another aspect of thepresent invention there is provided a pharmaceutical composition whichincludes, as an active ingredient thereof, a protein and apharmaceutical acceptable carrier. As used herein a “pharmaceuticalcomposition” refers to a preparation of one or more of the activeingredients described herein, such as a recombinant protein, with otherchemical components such as traditional drugs, physiologically suitablecarriers and excipients. The purpose of a pharmaceutical composition isto facilitate administration of a protein or cell to an organism.Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

In a preferred embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Hereinafter, the phrases “physiologically suitable carrier” and“pharmaceutically acceptable carrier” are interchangeably used and referto an approved carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered conjugate.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of theprotein, preferably in purified form, together with a suitable amount ofcarrier so as to provide the form for proper administration to thepatient. The formulation should be suitable for the mode ofadministration.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate processes andadministration of the active ingredients. Examples, without limitation,of excipients include calcium carbonate, calcium phosphate, varioussugars and types of starch, cellulose derivatives, gelatin, vegetableoils and polyethylene glycols.

Further techniques for formulation and administration of activeingredients may be found in “Remington's Pharmaceutical Sciences,” MackPublishing Co., Easton, Pa., latest edition, which is incorporatedherein by reference as if fully set forth herein.

The pharmaceutical compositions herein described may also comprisesuitable solid or gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, intestinal or parenteral delivery,including intramuscular, subcutaneous and intramedullary injections aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants are used in theformulation. Such penetrants are generally known in the art.

For oral administration, the active ingredients can be optionallyformulated through administration of the whole cells producing a proteinaccording to the present invention, such as GCD for example. The activeingredients can also be formulated by combining the active ingredientsand/or the cells with pharmaceutically acceptable carriers well known inthe art. Such carriers enable the active ingredients of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions, and the like, for oral ingestion by apatient. Pharmacological preparations for oral use can be made using asolid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries ifdesired, to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active ingredient doses.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the active ingredient and a suitable powderbase such as lactose or starch.

The active ingredients described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidsesters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theactive ingredients to allow for the preparation of highly concentratedsolutions.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, pharmaceuticalcompositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The pharmaceutical compositions of the invention can be formulated asneutral or salt forms. Pharmaceutically acceptable salts include thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The active ingredients of the present invention may also be formulatedin rectal compositions such as suppositories or retention enemas, using,e.g., conventional suppository bases such as cocoa butter or otherglycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

The topical route is optionally performed, and is assisted by a topicalcarrier. The topical carrier is one which is generally suited fortopical active ingredient administration and includes any such materialsknown in the art. The topical carrier is selected so as to provide thecomposition in the desired form, e.g., as a liquid or non-liquidcarrier, lotion, cream, paste, gel, powder, ointment, solvent, liquiddiluent, drops and the like, and may be comprised of a material ofeither naturally occurring or synthetic origin. It is essential,clearly, that the selected carrier does not adversely affect the activeagent or other components of the topical formulation, and which isstable with respect to all components of the topical formulation.Examples of suitable topical carriers for use herein include water,alcohols and other nontoxic organic solvents, glycerin, mineral oil,silicone, petroleum jelly, lanolin, fatty acids, vegetable oils,parabens, waxes, and the like. Preferred formulations herein arecolorless, odorless ointments, liquids, lotions, creams and gels.

Ointments are semisolid preparations, which are typically based onpetrolatum or other petroleum derivatives. The specific ointment base tobe used, as will be appreciated by those skilled in the art, is one thatwill provide for optimum active ingredients delivery, and, preferably,will provide for other desired characteristics as well, e.g., emolliencyor the like. As with other carriers or vehicles, an ointment base shouldbe inert, stable, nonirritating and nonsensitizing. As explained inRemington: The Science and Practice of Pharmacy, 19th Ed. (Easton, Pa.:Mack Publishing Co., 1995), at pages 1399-1404, ointment bases may begrouped in four classes: oleaginous bases; emulsifiable bases; emulsionbases; and water-soluble bases. Oleaginous ointment bases include, forexample, vegetable oils, fats obtained from animals, and semisolidhydrocarbons obtained from petroleum. Emulsifiable ointment bases, alsoknown as absorbent ointment bases, contain little or no water andinclude, for example, hydroxystearin sulfate, anhydrous lanolin andhydrophilic petrolatum. Emulsion ointment bases are either water-in-oil(W/O) emulsions or oil-in-water (O/W) emulsions, and include, forexample, cetyl alcohol, glyceryl monostearate, lanolin and stearic acid.Preferred water-soluble ointment bases are prepared from polyethyleneglycols of varying molecular weight; again, reference may be made toRemington: The Science and Practice of Pharmacy for further information.

Lotions are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semiliquid preparations, in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and maycomprise a liquid oily emulsion of the oil-in-water type. Lotions arepreferred formulations herein for treating large body areas, because ofthe ease of applying a more fluid composition. It is generally necessarythat the insoluble matter in a lotion be finely divided. Lotions willtypically contain suspending agents to produce better dispersions aswell as active ingredients useful for localizing and holding the activeagent in contact with the skin, e.g., methylcellulose, sodiumcarboxymethylcellulose, or the like.

Creams containing the selected active ingredients are, as known in theart, viscous liquid or semisolid emulsions, either oil-in-water orwater-in-oil. Cream bases are water-washable, and contain an oil phase,an emulsifier and an aqueous phase. The oil phase, also sometimes calledthe “internal” phase, is generally comprised of petrolatum and a fattyalcohol such as cetyl or stearyl alcohol; the aqueous phase usually,although not necessarily, exceeds the oil phase in volume, and generallycontains a humectant. The emulsifier in a cream formulation, asexplained in Remington, supra, is generally a nonionic, anionic,cationic or amphoteric surfactant.

Gel formulations are preferred for application to the scalp. As will beappreciated by those working in the field of topical active ingredientsformulation, gels are semisolid, suspension-type systems. Single-phasegels contain organic macromolecules distributed substantially uniformlythroughout the carrier liquid, which is typically aqueous, but also,preferably, contain an alcohol and, optionally, an oil.

Various additives, known to those skilled in the art, may be included inthe topical formulations of the invention. For example, solvents may beused to solubilize certain active ingredients substances. Other optionaladditives include skin permeation enhancers, opacifiers, anti-oxidants,gelling agents, thickening agents, stabilizers, and the like.

The topical compositions of the present invention may also be deliveredto the skin using conventional dermal-type patches or articles, whereinthe active ingredients composition is contained within a laminatedstructure, that serves as a drug delivery device to be affixed to theskin. In such a structure, the active ingredients composition iscontained in a layer, or “reservoir”, underlying an upper backing layer.The laminated structure may contain a single reservoir, or it maycontain multiple reservoirs. In one embodiment, the reservoir comprisesa polymeric matrix of a pharmaceutically acceptable contact adhesivematerial that serves to affix the system to the skin during activeingredients delivery. Examples of suitable skin contact adhesivematerials include, but are not limited to, polyethylenes, polysiloxanes,polyisobutylenes, polyacrylates, polyurethanes, and the like. Theparticular polymeric adhesive selected will depend on the particularactive ingredients, vehicle, etc., i.e., the adhesive must be compatiblewith all components of the active ingredients-containing composition.Alternatively, the active ingredients-containing reservoir and skincontact adhesive are present as separate and distinct layers, with theadhesive underlying the reservoir which, in this case, may be either apolymeric matrix as described above, or it may be a liquid or hydrogelreservoir, or may take some other form.

The backing layer in these laminates, which serves as the upper surfaceof the device, functions as the primary structural element of thelaminated structure and provides the device with much of itsflexibility. The material selected for the backing material should beselected so that it is substantially impermeable to the activeingredients and to any other components of the activeingredients-containing composition, thus preventing loss of anycomponents through the upper surface of the device. The backing layermay be either occlusive or non-occlusive, depending on whether it isdesired that the skin become hydrated during active ingredientsdelivery. The backing is preferably made of a sheet or film of apreferably flexible elastomeric material. Examples of polymers that aresuitable for the backing layer include polyethylene, polypropylene, andpolyesters.

During storage and prior to use, the laminated structure includes arelease liner. Immediately prior to use, this layer is removed from thedevice to expose the basal surface thereof, either the activeingredients reservoir or a separate contact adhesive layer, so that thesystem may be affixed to the skin. The release liner should be made froman active ingredients/vehicle impermeable material.

Such devices may be fabricated using conventional techniques, known inthe art, for example by casting a fluid admixture of adhesive, activeingredients and vehicle onto the backing layer, followed by laminationof the release liner. Similarly, the adhesive mixture may be cast ontothe release liner, followed by lamination of the backing layer.Alternatively, the active ingredients reservoir may be prepared in theabsence of active ingredients or excipient, and then loaded by “soaking”in an active ingredients/vehicle mixture.

As with the topical formulations of the invention, the activeingredients composition contained within the active ingredientsreservoirs of these laminated system may contain a number of components.In some cases, the active ingredients may be delivered “neat,” i.e., inthe absence of additional liquid. In most cases, however, the activeingredients will be dissolved, dispersed or suspended in a suitablepharmaceutically acceptable vehicle, typically a solvent or gel. Othercomponents, which may be present, include preservatives, stabilizers,surfactants, and the like.

It should be noted that the protein of the invention, such as a highmannose lysosomal enzyme, is preferably administered to the patient inneed in an effective amount. As used herein, “effective amount” means anamount necessary to achieve a selected result. For example, an effectiveamount of the composition of the invention may be selected for beinguseful for the treatment of a lysosomal storage disease.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredient effective to prevent, alleviate or ameliorate symptomsof disease or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any active ingredient used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromactivity assays in animals. For example, a dose can be formulated inanimal models to achieve a circulating concentration range that includesthe IC₅₀ as determined by activity assays.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures inexperimental animals, e.g., by determining the IC₅₀ and the LD₅₀ (lethaldose causing death in 50% of the tested animals) for a subject activeingredient. The data obtained from these activity assays and animalstudies can be used in formulating a range of dosage for use in human.For example, therapeutically effective doses suitable for treatment ofgenetic disorders can be determined from the experiments with animalmodels of these diseases.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, termed the minimal effective concentration (MEC).The MEC will vary for each preparation, but may optionally be estimatedfrom whole animal data.

Dosage intervals can also be determined using the MEC value.Preparations may optionally be administered using a regimen, whichmaintains plasma levels above the MEC for 10-90% of the time, preferablebetween 30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the condition to betreated, dosing can also be a single administration of a slow releasecomposition described hereinabove, with course of treatment lasting fromseveral days to several weeks or until cure is effected or diminution ofthe disease state is achieved.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accompanied by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising an active ingredient of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

As used herein, the term “modulate” includes substantially inhibiting,slowing or reversing the progression of a disease, substantiallyameliorating clinical symptoms of a disease or condition, orsubstantially preventing the appearance of clinical symptoms of adisease or condition. A “modulator” therefore includes an agent whichmay modulate a disease or condition.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

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1. An isolated human glucocerebrosidase protein having the amino acidsequence as set forth in SEQ ID NO:
 15. 2. A pharmaceutical compositioncomprising the glucocerebrosidase protein of claim 1 and apharmaceutically acceptable carrier.
 3. The human glucocerebrosidaseprotein of claim 1, being glycosylated with at least one xylose and atleast one exposed mannose residue.
 4. The human glucocerebrosidaseprotein of claim 1, being glycosylated with at least one core α-(1,2)xylose and at least one core α-(1,3) fucose.
 5. The humanglucocerebrosidase protein of claim 4, being glycosylated with at leastone exposed mannose residue.
 6. The human glucocerebrosidase protein ofclaim 1, being glycosylated with at least one exposed mannose residueand at least one fucose residue having an alpha (1-3) glycosidic bond.7. The human glucocerebrosidase protein of claim 3, wherein said xyloseresidue is a core α-(1,2) xylose residue.
 8. The humanglucocerebrosidase protein of claim 3, wherein said glucocerebrosidaseprotein binds to a mannose receptor on a macrophage.
 9. The humanglucocerebrosidase protein of claim 5, wherein said glucocerebrosidaseprotein binds to a mannose receptor on a macrophage.
 10. The humanglucocerebrosidase protein of claim 8, wherein said glucocerebrosidaseis taken up into macrophages.
 11. The human glucocerebrosidase proteinof claim 1 wherein said glucocerebrosidase protein has enzymaticactivity.
 12. The human glucocerebrosidase protein of claim 3 whereinsaid glucocerebrosidase protein has enzymatic activity.
 13. The humanglucocerebrosidase protein of claim 8, having an increased uptake bysaid macrophages, in comparison with the corresponding macrophage uptakeof a recombinant glucocerebrosidase expressed in mammalian cells.
 14. Apharmaceutical composition comprising the human glucocerebrosidaseprotein of claim 3 and a pharmaceutically acceptable carrier.
 15. Apharmaceutical composition comprising the human glucocerebrosidaseprotein of claim 5 and a pharmaceutically acceptable carrier.
 16. Aplant cell preparation comprising the human glucocerebrosidase proteinof claim
 1. 17. A plant cell preparation comprising the humanglucocerebrosidase protein of claim
 3. 18. A plant cell preparationcomprising the human glucocerebrosidase protein of claim
 5. 19. Theplant cell preparation of claim 17, wherein said humanglucocerebrosidase protein having at least one exposed mannose residuecomprises a dominant fraction of said glucocerebrosidase protein, asmeasured by linkage analysis.
 20. A pharmaceutical compositioncomprising the plant cell preparation of claim 16 and a pharmaceuticallyacceptable carrier.
 21. A method for treating a subject having Gaucher'sdisease using exogenous human glucocerebrosidase protein, the methodcomprising: (a) providing the glucocerebrosidase protein of claim 3; and(b) administering a therapeutically effective amount of saidglucocerebrosidase protein to said subject.
 22. A method for treating asubject having Gaucher's disease using exogenous humanglucocerebrosidase protein, the method comprising: (a) providing theglucocerebrosidase protein of claim 5; and (b) administering atherapeutically effective amount of said glucocerebrosidase protein saidsubject.